Phosphoramidite building blocks for sugar-conjugated oligonucleotides

ABSTRACT

Novel nucleoside phosphoramidite building blocks for preparation of synthetic oligonucleotides containing at least one phosphotriester linkage conjugated to a monosaccharide and synthetic processes for making the same are disclosed. Furthermore, oligomeric compounds are prepared using said building blocks, preferably followed by removal of protecting groups to provide monosaccharide-conjugated oligonucleotides.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 14/960,197, filed Dec. 4, 2015, which is a continuationapplication of PCT Application No. PCT/US2014/041133, filed on Jun. 5,2014, which claims the benefit of priority of U.S. ProvisionalApplication No. 61/831,521, filed on Jun. 5, 2013. Each of the foregoingapplications is hereby incorporated by reference as though set forthherein in its entirety.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED AS AN ASCII FILE

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference herein inits entirety. Said ASCII copy, created on Aug. 3, 2018, is named“095111-1099045_ST25.txt” and is 23,775 bytes in size.

BACKGROUND OF THE INVENTION 1. Field of the Invention

In one aspect, this invention is directed to methods for the preparationof oligonucleotides conjugated to at least one sugar moiety. In theother aspect, the invention provides methods for preparation ofprotected forms of oligonucleotides wherein at least one of theinternucleosidic phosphate moieties is converted to its phosphatetriester analog and to compounds useful in preparation of such protectedoligonucleotides. Oligonucleotides prepared by the methods of theinvention are useful for therapeutic and diagnostic applications and asresearch reagents.

2. Summary of the Related Art

It is well known, however, that oligonucleotides and theirphosphorothioate analogues are of limited stability in blood andtissues. Also, since such compounds are negatively charged they lack theability to efficiently permeate biological membranes. Thus, both theiroral bioavailability and cellular uptake are usually low. To overcomethis problem, several types of modified oligonucleotides have beenintroduced. Among such oligonucleotides, backbone modified neutraloligonucleotides, phosphate triester analogs, have gained widerecognition.

To date, the art recognizes simple alkyl triester analogs, methylphosphotriester analogs disclosed in U.S. Pat. No. 5,955,599, and ethylphosphotriester analogs disclosed in Pless, R. C.; Ts'o, P. O. P.Biochemistry 1977, 16 (6), 1239-1250; Miller, P. S.; Barrett, J. C.;Ts'o, P. O. P. Biochemistry 1974, 13 (24), 4887-96; Lesser, D. R.; etal. J Biol. Chem. 1992, 267 (34), 24810-18; and Stawinski, J.; et al.Tetrahedron Lett. 1992, 33 (22), 3185-8. More complex, O-acyloxyaryltriester analogs have been disclosed in Iyer, R. P.; et al. Bioorg. Med.Chem. Lett. 1997, 7 (7), 871-876. Further development was presented byacylaminoethyl analogs disclosed in U.S. Pat. No. 6,121,437, U.S. Pat.No. 6,610,837, US 2001/0044529, WO 2003/048179, and WO 2006/065751.(Pivaloylthio)ethyl triester analogs have been disclosed in U.S. Pat.No. 6,919,437. The latter modification has been even further developedwith the introduction of extended side chains to replace the ethylfragment as disclosed in WO2010/039543 and in WO2014/031575. Severalother, less developed, embodiments of triester analogs have beenreported in Sekine, M.; et al. Eur. J. Org. Chem. 2001, (10), 1989-1999;Sekine, M.; et al. J. Org. Chem. 2000, 65 (20), 6515-6524; Sobkowski,M.; et al. Tetrahedron Lett. 1995, 36 (13), 2295-8; Ayukawa, H.; et al.Chemistry Lett. 1995, (1), 81.

The common feature of the analogs disclosed in the prior art was thatthe elimination of negative charge from the backbone of modifiedoligonucleotides led to the loss of solubility of said oligonucleotidesin aqueous media, which is highly desirable for their successful use inbiological applications.

SUMMARY OF THE INVENTION

Those skilled in the art will appreciate the fact that naturalcarbohydrates, particularly mono- and oligosaccharides, arecharacterized by a high degree of solubility in aqueous media. Shouldmono- or oligosaccharides be artificially conjugated to compounds of lowsolubility, this property may be used for the benefit of dissolution ofthe compounds in aqueous systems.

Several processes for the solid phase synthesis of oligonucleotidecompounds are known to those skilled in the art and may be employed withthe present invention. Exemplary processes are disclosed in U.S. Pat.No. 4,458,066 issued on Jul. 3, 1984, U.S. Pat. No. 4,500,707 issued onFeb. 19, 1985, and U.S. Pat. No. 5,132,418 issued on Nov. 27, 1990.

A process for the preparation of phosphoramidite building blocks isdisclosed in U.S. Pat. No. 4,415,732 issued on Nov. 15, 1983. Certainnucleoside phosphoramidite compounds are disclosed in U.S. Pat. No.4,668,777 issued on May 26, 1987.

It is an object of this invention to provide novel compounds which mayserve as building blocks for the preparation of oligomeric compounds,phosphotriester analogs of natural oligonucleotides, wherein acarbohydrate moiety is linked to the internucleosidic phosphate residue.

It is a further object of the present invention to provide noveloligomeric compounds, phosphotriester analogs of naturaloligonucleotides with improved physico-chemical properties, wherein acarbohydrate moiety is linked to the internucleosidic phosphate residue.

It is a further object of this invention to provide methods forsynthetic preparation of said oligomeric compounds.

Other objects of this invention will be apparent to those skilled in theart.

These objects are satisfied by the present invention which providesnovel nucleoside phosphoramidite reagents useful in preparation ofoligomeric compounds and methods for making such oligomeric compounds.

Abbreviations

As used herein:

-   -   Ac is Acetyl;    -   Bz is benzoyl;    -   DCM is dichloromethane;    -   dmf is N,N-dimethylformamidino;    -   iPrPac is (4-isopropylphenoxy)acetyl;    -   MeCN is acetonitrile;    -   Pac is phenoxyacetyl;    -   TEA is triethylamine;    -   NMI is N-methylimidazol;    -   HPLC is high-performance liquid chromatography; and    -   ES-MS is mass-spectrometry with electron-spray ionization.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows structures of compounds 1, 2, and 3 and a synthetic schemefor the preparation of compounds 7a-7e.

FIG. 2 shows a synthetic scheme for the preparation of compounds 12,13a-13c, 14a-14d, and 15a-15d.

FIG. 3 shows a synthetic scheme for the preparation of compounds 16,17a-17c, 18a-18d, and 19a-19d.

FIG. 4 shows a synthetic scheme for the preparation of compounds 20,21a, 21c, 22a, 22b, 23a, and 23b.

FIG. 5 shows a synthetic scheme for the preparation of compounds 24,25a, 25c, 26a, 26b, 27a, and 27b.

FIG. 6 shows a synthetic scheme for the preparation of compounds 28,29a, 29c, 30a, 30b, 31a, 31b, 32, 33a, 33c, 34a, 34b, 35a, 35b, 36,37a-37c, 38a-38d, 39a-39d, 40, 41a, 41c, 42a, 42b, 43a, and 43b.

FIG. 7 shows a synthetic scheme for the preparation of compounds 52,53a, 53c, 54a, 54b, 55a, 55b, 56, 57a, 57c, 58a, 58b, 59a, and 59b.

FIG. 8 shows an alternative synthetic scheme for the preparation ofcompound 28.

FIG. 9 shows phosphotriester nucleoside residues 65, 66, 75, 76, 77, 78,79, and 80 incorporated into synthetic oligonucleotides.

FIG. 10 shows an exemplary ³¹P NMR spectrum of5′-O-(4,4′-dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[11-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6,9-trioxaundecyloxy]phosphinyl]thymidine(36, Example 57).

DETAILED DESCRIPTION OF THE INVENTION

In a first aspect, the invention provides novel compounds which mayserve as building blocks for preparation of oligomeric compounds,phosphotriester analogs of natural oligonucleotides, wherein acarbohydrate moiety is linked to the internucleosidic phosphate residueaccording to Formula I:

wherein:

-   -   R¹ is an optionally protected nucleic base selected from        adenine, cytosine, guanine, thymine, uracil, 2-aminoadenine,        N6-methyladenine, 7-deazaadenine, 7-deaza-8-azaadenine,        8-aminoadenine, 5-methylcytosine, N4-ethylcytosine,        7-deazaguanine, 7-deaza-8-azaguanine, 8-aminoguanine,        7-deazaxanthyne, or hypoxanthine;    -   one of R² and R^(2a) is a protecting group of trityl type        including but not limited to (4-methoxyphenyl)diphenylmethyl,        bis-(4-methoxyphenyl)phenylmethyl, tris-(methoxyphenyl)methyl,        9-phenylxanthen-9-yl, or 9-(p-methoxyphenyl)xanthen-9-yl, and        the other is a phosphoramidite moiety:

wherein:

-   -   each R⁴ and R⁵ is, independently, C₁ to C₆ alkyl, or R⁴ and R⁵        together with the nitrogen atom they are attached form a cycle        wherein R⁴+R⁵═(CH₂)_(n)X(CH₂)_(m),    -   wherein:        -   X is an atom of oxygen or CH₂ group;        -   each n and m is, independently, an integer from 2 to about            5;    -   L is a linking moiety —[(CH₂)_(p)Y(CH₂)_(q)]_(r)—,    -   wherein:        -   each p, q, and r is, independently, an integer from 1 to 18;        -   Y is a chemical bond, oxygen atom, sulfur atom, NQ¹,            —N(Q¹)C(═O)N(Q²)-, —C(═O)N(Q¹)-, or —N(Q¹)C(═O)—,    -   wherein:        -   each Q¹ and Q² is independently hydrogen atom, methyl group,            ethyl group, propyl group, isopropyl group, acetyl group,            trifluoroacetyl group, phenoxyacetyl group, benzoyl group,            or 9-fluorenylmethyloxycarbonyl group;    -   R⁶ is a substituted monosaccharide residue of general Formula II

wherein:

-   -   each R¹⁰ is, independently, an acyl protecting group including        but not limited to acetyl, propionyl, butyryl, isobutyryl,        pivaloyl, benzoyl, or 4-methylbenzoyl groups, a trityl-type        protecting group including but not limited to        (4-methoxyphenyl)diphenylmethyl,        bis-(4-methoxyphenyl)phenylmethyl, tris-(methoxyphenyl)methyl,        9-phenylxanthen-9-yl, or 9-(p-methoxyphenyl)xanthen-9-yl, a        silyl protecting group including but not limited to        triisopropylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl,        triphenylsilyl, or diphenylmethylsilyl, an alkyl group        containing from 1 to 18 atoms of carbon, a benzyl group, a        4-methoxybenzyl group, a propargyl group, or another substituted        monosacharide residue of Formula II;    -   A is a chemical bond or CHOR¹⁰;    -   Z is a hydrogen, OR¹⁰, or N(Q²)Q³ wherein:        -   each Q² and Q³ is, independently, hydrogen atom, methyl            group, ethyl group, propyl group, isopropyl group, acetyl            group, trifluoroacetyl group, phenoxyacetyl group, benzoyl            group, or 9-fluorenylmethyloxycarbonyl group;

-   R³ is hydrogen atom, fluorine atom, substituted hydroxy group OR⁷,    or substituted amino group NR⁸R⁹,    wherein:    -   R⁷ is a C₁ to C₆ alkyl, 2-alkoxyethyl group, or        N-methylcarboxamidomethyl group; and    -   each R⁸ and R⁹ is, independently, hydrogen atom, methyl group,        ethyl group, propyl group, isopropyl group, acetyl group,        trifluoroacetyl group, phenoxyacetyl group, benzoyl group, or        9-fluorenylmethyloxycarbonyl group.

In an embodiment of the present invention, R¹ of Formula I is a nucleicbase selected from N⁶-benzoyladenine, N⁶-phenoxyacetyladenine,N⁶-(4-isopropylphenoxy)acetyladenine, adenine, N⁴-benzoylcytosine,N⁴-acetylcytosine, N⁴-phenoxyacetylcytosine,N⁴-(4-isopropylphenoxy)acetylcytosine, N⁴-benzoyl-4-methylcytosine,N⁴-acetyl-4-methylcytosine, cytosine, N⁴-phenoxyacetyl-4-methylcytosine,N⁴-(4-isopropylphenoxy)acetyl-4-methylcytosine, 4-methylcytosine,N²-isobutyrylguanine, N²-phenoxyacetylguanine,N²-(4-isopropylphenoxy)acetylguanine,N²—(N,N-dimethylformamidino)guanine, guanine, thymine, or uracil.

In another embodiment, R² of Formula I is 4,4′-dimethoxytrityl group andR^(2a) is

In yet another embodiment, R^(2a) of Formula I is 4,4′-dimethoxytritylgroup and R² is

In yet another embodiment of the present invention, R³ of Formula I ishydrogen.

In yet another embodiment of the present invention, R³ of Formula I isOCH₃.

In yet another embodiment of the present invention, R³ of Formula I isfluorine.

In yet another embodiment of the present invention, each R⁴ and R⁵ ofFormula I is isopropyl group.

In yet another embodiment of the present invention, L of Formula I is—(CH₂)₃—.

In yet another embodiment of the present invention, L of Formula I is—(CH₂)₄—.

In yet another embodiment of the present invention, L-O of Formula I is—[(CH₂)₂—O]₂—.

In yet another embodiment of the present invention, L-O of Formula I is—[(CH₂)₂—O]₃—.

In yet another embodiment of the present invention, L-O of Formula I is—[(CH₂)₂—O]₄—.

In yet another embodiment of the present invention, L-O of Formula I is—[(CH₂)₂—O]₅—.

In yet another embodiment of the present invention, R⁶ of Formula I is aprotected β-D-glucopyranoside.

In yet another embodiment of the present invention, R⁶ of Formula I is aprotected β-D-galactopyranoside.

In yet another embodiment of the present invention, R⁶ of Formula I is aprotected 2-amino-2-deoxy-β-D-glucopyranoside.

In still another embodiment of the present invention, R⁶ of Formula I isa protected 2-amino-2-deoxy-β-D-glucopyranoside wherein one of Q² and Q³is hydrogen, and the other is an acetyl group.

In a further embodiment of the present invention, R⁶ of Formula I is aprotected 2-amino-2-deoxy-β-D-galactopyranoside.

In a still further embodiment of the present invention, R⁶ of Formula Iis a protected 2-amino-2-deoxy-β-D-galactopyranoside wherein one of Q²and Q³ is hydrogen, and the other is an acetyl group.

In yet another embodiment of the present invention, each R¹⁰ of FormulaII is an acetyl group.

In yet another embodiment of the present invention, each R¹⁰ of FormulaII is a benzoyl group.

In yet another embodiment of the present invention, each R¹⁰ of FormulaII is a butyryl group.

In yet another embodiment of the present invention, each R¹⁰ of FormulaII is an isobutyryl group.

In yet another embodiment of the present invention, each R¹⁰ of FormulaII is a propionyl group.

In yet another embodiment of the present invention, each R¹⁰ of FormulaII is a 4-methylbenzoyl group.

In yet another preferred embodiment of the present invention, one of R¹⁰of Formula II is another protected monosaccharide residue of Formula II,and each of the other R¹⁰ is an acetyl group.

In a second aspect, the present invention provides novel oligomericcompounds, phosphotriester analogs of natural oligonucleotides, havingthe structure according to Formula III wherein a carbohydrate moiety islinked to the internucleosidic phosphate residue:

wherein:

-   -   each R¹ is independently an optionally protected nucleic base        selected from adenine, cytosine, guanine, thymine, uracil,        2-aminoadenine, N6-methyladenine, 7-deazaadenine,        7-deaza-8-azaadenine, 8-aminoadenine, 5-methylcytosine,        N4-ethylcytosine, 7-deazaguanine, 7-deaza-8-azaguanine,        8-aminoguanine, 7-deazaxanthyne, or hypoxanthine;    -   each R³ is, independently, hydrogen atom, fluorine atom,        substituted hydroxy group OR⁷, or substituted amino group NR⁸R⁹,    -   wherein:        -   each R⁷ is, independently, a C₁ to C₆ alkyl, 2-alkoxyethyl            group, or N-methylcarboxamidomethyl group;        -   each R⁸ and R⁹ is, independently, hydrogen atom, methyl            group, ethyl group, propyl group, isopropyl group, acetyl            group, trifluoroacetyl group, phenoxyacetyl group, benzoyl            group, or 9-fluorenylmethyloxycarbonyl group;    -   L is a linking moiety —[(CH₂)_(p)Y(CH₂)_(q)]_(r)—,    -   wherein:        -   each p, q, and r is, independently, an integer from 1 to 18;        -   Y is a chemical bond, oxygen atom, sulfur atom, NQ¹,            —N(Q¹)C(═O)N(Q²)-, —C(═O)N(Q¹)-, or —N(Q¹)C(═O)—,        -   wherein:            -   each Q¹ and Q² is independently hydrogen atom, methyl                group, ethyl group, propyl group, isopropyl group,                acetyl group, trifluoroacetyl group, phenoxyacetyl                group, benzoyl group, or 9-fluorenylmethyloxycarbonyl                group;    -   each R⁶ is, independently, a substituted monosaccharide residue        of general Formula II,    -   wherein:        -   each R¹⁰ is, independently, an acyl protecting group            including but not limited to acetyl, propionyl, butyryl,            isobutyryl, pivaloyl, benzoyl, and 4-methylbenzoyl groups, a            trityl-type protecting group including but not limited to            (4-methoxyphenyl)diphenylmethyl,            bis-(4-methoxyphenyl)phenylmethyl,            tris-(methoxyphenyl)methyl, 9-phenylxanthen-9-yl, and            9-(p-methoxyphenyl)xanthen-9-yl, a silyl protecting group            including but not limited to triisopropylsilyl,            t-butyldimethylsilyl, t-butyldiphenylsilyl, triphenylsilyl,            and diphenylmethylsilyl, an alkyl group containing from 1 to            18 atoms of carbon, a benzyl group, a 4-methoxybenzyl group,            a propargyl group, or another substituted monosacharide            residue of Formula II;        -   A is a chemical bond or CHOR¹⁰;        -   Z is a hydrogen, OR¹⁰, or N(Q²)Q³ wherein:            -   each Q² and Q³ is, independently, hydrogen atom, methyl                group, ethyl group, propyl group, isopropyl group,                acetyl group, trifluoroacetyl group, phenoxyacetyl                group, benzoyl group, or 9-fluorenylmethyloxycarbonyl                group;    -   each R¹¹ is independently a negative charge compensated by a        cation, a phosphate protecting group, or R⁶O-L-;    -   each R¹² and R¹³ is, independently, hydrogen atom, a protecting        group selected from (4-methoxyphenyl)diphenylmethyl,        bis-(4-methoxyphenyl)phenylmethyl, tris-(methoxyphenyl)methyl,        9-phenylxanthen-9-yl, or 9-(p-methoxyphenyl)xanthen-9-yl; or a        point of attachment to solid phase material with the proviso        that R¹² and R¹³ are not both simultaneously a linker connected        to a solid support;    -   each W¹, W², and W³ is independently oxygen or sulfur; and    -   each s and t is, independently, an integer from 0 to about 100.

In a third aspect, the present invention provides methods for syntheticpreparation of oligomeric compounds according to Formula III, saidmethod comprising:

-   -   reacting a compound of Formula I with a compound of Formula IV        containing at least one reactive hydroxy group wherein the        compound of Formula IV has the structure:

-   -   wherein:        -   one of R¹² and R¹³ is a hydrogen atom, and the other is a            protecting group or a linker connected to a solid support;            and        -   k is an integer selected from 0 to about 100.

In an embodiment, the method further comprises treating the oligomericcompound with a reagent under conditions of time, temperature, andpressure effective to oxidize or sulfurize the oligomeric compound.

In an embodiment, the method further comprises treating the oligomericcompound with a reagent under conditions of time, temperature, andpressure effective to deprotect the oligomeric compound.

In an embodiment, wherein R¹³ is a linker connected to a solid support,the method further comprises treating the oligomeric compound with areagent under conditions of time, temperature, and pressure effective toremove the oligomeric compound from the solid support.

Certain of the starting materials used in the practice of the presentinvention are protected nucleosides 8, 9a-9c, 10a-10d, and 1a-11d, whichare readily available from commercial sources (such as, for example,ChemGenes, Inc., Waltham, Mass.; Rasayan, Inc., Encinitas, Calif.).

Certain other starting materials used in the practice of the presentinvention are readily prepared following methods and procedures familiarto those skilled in the art and disclosed in the chemical literature.Compounds 1, 2, and 3 (FIG. 1) were prepared as disclosed in Karjala, S.and Link, K. P., J. Amer. Chem. Soc., 1940, 62, 917-922. Compound 4(FIG. 1) was prepared as disclosed in Fukase, K; et al. Bull. Chem. Soc.Japan 2003, 76, 485-500, and compound 5 (FIG. 1) was prepared asdisclosed in Matta, K. L.; et al. Carbohydrate Res. 1973, 26, 215-218.

Compounds 6b and 7a-7e were prepared as disclosed in FIG. 1 fromcompounds 4 and 5 and ethyleneglycol, di- tri- tetra- andpentaethyleneglycols.

As illustrated in FIG. 2, exemplary phosphoramidite building blocks 12,13a-13c, 14a-14d, 15a-15d comprising a protected β-D-glucopyranosemoiety attached to the phosphite function via a C₃-spacer arm may bereadily synthesized from protected 2′-deoxynucleosides 8, 9a-9c,10a-10d, and 11a-11d and 3-hydroxypropyl2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside 1.

To synthesize compounds 12, 13a-13c, 14a-14d, 15a-15d, the respectiveprotected 2′-deoxynucleoside 8, 9a-9c, 10a-10d, and 11a-11d was firsttreated with bis(N,N-diisopropylamino) chlorophosphite (R. I. Chemical,Orange, Calif.) in the presence of excess N-ethyl-N,N-diisopropylamine(Alfa Aesar, Ward Hill, Mass.) in MeCN at −20° C. followed by stirringat room temperature for 1 to 2 h. The obtained solution of protectednucleoside-3′-O-bis(N,N-diisopropylamino) phosphite was further treatedwith compound 1 and 1H-tetrazole (Glen Research, Sterling, Va.) for 14 hat room temperature. Quenching with 5% aqueous NaHCO₃ followed by thestandard work-up and purification by chromatography on a silica gelcolumn afforded the desired phosphoramidite building blocks 12, 13a-13c,14a-14d, 15a-15d.

Among these compounds, phosphoramidite building blocks 13a, 14a, and 15afeature the standard protecting scheme for exocyclic amino groups ofnucleic bases.

The method described above is also applicable to a peracetylatedβ-D-glucopyranoside-bearing ethyleneglycol spacer arm. FIG. 3illustrates a reaction of protected nucleosides 8, 9a-9c, 10a-10d, and11a-11d with 2-[(2-hydroxyethyl)oxy]ethyl2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside 2. Following the proceduredescribed above gave the phosphoramidite building blocks 16, 17a-17c,18a-18d, 19a-19d featuring a diethyleneglycol spacer arm.

In certain embodiments, it is desirable to introduce monosaccharideresidues other than glucose into oligonucleotides. By following thegeneral synthetic method described above, the preparation ofphosphoramidite building blocks 20, 21a, 21c, 22a, 22b, 23a, and 23bderivatized with a peracetylated β-D-galactospyranoside residue andethyleneglycol spacer arm was accomplished by reacting protectednucleosides 8, 9a-9c, 10a-10d, 11a-11d with compound 3 (see FIG. 4).

By following the general synthetic method described above, thepreparation of phosphoramidite building blocks 24, 25a, 25c, 26a, 26b,27a, and 27b derivatized with a protected N-acetyl-D-glucosamine residueand C3 spacer arm was accomplished by reacting protected nucleosides 8,9a-9c, 10a-10d, 1a-1d with compound 6 (see FIG. 5).

Analogously, the preparation of phosphoramidite building blocks 28, 29a,29c, 30a, 30b, 31a, 31b, 32, 33a, 33c, 34a, 34b, 35a, 35b, 36, 37a-37c,38a-38d, 39a-39d, 40, 41a, 41c, 42a, 42b, 43a, and 43b derivatized withprotected N-acetyl-D-galactosamine residues starting from protectednucleosides 8, 9a-9c, 10a-10d, and 11a-11d and compounds 7a-7e isdescribed in FIG. 6. The phosphoramidite reagents of this series featureoligoethyleneglycol linkers of various lengths to allow the placement ofthe monosaccharide residue at different distances from polyphosphatebackbone of oligonucleotides.

In certain embodiments of the invention, synthetic oligonucleotidesbearing nucleotide residues derivatized at the 2′-position of the ribosering are desirable due to their enhanced base-pairing properties. Thoseskilled in the art recognize that 2′-OMe and 2′-fluoro substitution atthe ribose ring results in the enhancement of hybridization affinity ofderivatized oligonucleotides to the complementary strands ofoligonucleotides or nucleic acids. FIG. 7 describes the preparation ofexemplary phosphoramidite building blocks 52, 53a, 53c, 54a, 54b, 55a,and 55b derived from 2′-O-methylnucleosides 44, 45a, 45c, 46a, 46b, 47a,47b and phosphoramidite building blocks 56, 57a, 57c, 58a, 58b, 59a, and59b derived from 2′deoxy-2′-fluoronucleosides 48, 49a, 49c, 50a, 50b,51a, and 51b. All compounds 52-59 comprise a peracetylatedD-galactosamine residue and tetraethyleneglycol spacer arm.

It is well known in the art that the standard conditions ofoligonucleotide deprotection comprise treating a solid support-bound,protected oligonucleotide with 28-33% aqueous ammonium hydroxide for 8 hat 55° C. In certain embodiments of the present invention, compounds13a, 14a, 15a, 17a, 18a, 19a, 21a, 22a, 23a, 25a, 26a, 27a, 29a, 30a,31a, 33a, 34a, 35a, 41a, 42a, 43a, 53a, 54a, 55a, 57a, 58a, and 59a, aredesigned to be used within the standard deprotection strategy. However,certain oligonucleotides comprising unnatural nucleosidic residues maydemonstrate a rather low stability under the standard conditions.

Various protecting schemes have been developed in order to remove theprotecting groups under milder conditions. These works have beenexpertly reviewed in Beaucage, S. L. and Iyer, R. P. Tetrahedron 1992,48, 2223-2311 and Beaucage, S. L. and Iyer, R. P. Tetrahedron 1993, 49,6123-6194 and references referred to therein, all of which are hereinincorporated by reference. In order to satisfy the requirements ofalternative deprotection schemes, the building blocks bearing C^(Ac),A^(dmf), and G^(dmf) protected bases as, for instance, in compounds 13c,14d, and 15d, are compatible with mild deprotecting strategies ofoligonucleotide synthesis. The building blocks bearing C^(Pac), A^(Pac),A^(iPrPac), G^(Pac), and G^(iPrPac) protected bases as, for instance, incompounds 13b, 14b, 14c, 15b, and 15c were prepared to be compatiblewith ultra-mild deprotecting strategies.

The present disclosure is not limited by the methods described in FIGS.1-7. For example, an alternative method is exemplified in FIG. 8 by thepreparation of phosphoramidite building block 28. Treatment of compound7b with bis(N,N-diisopropylamino) chlorophosphite in the presence ofN-ethyl-N,N-diisopropylamine gave a phosphorodiamidite compound. Afterpurification on a silica gel column, the resulting compound was reactedwith protected nucleoside 8 to give the desired phosphoramidite buildingblock 28.

Most phosphoramidite building blocks described herein were isolated assolid foams or, in the case of compounds comprising tetraethyleneglycoland pentaethyleneglycol spacer, arms, as colorless oils sufficientlystable to be stored for several months at −18° C.

The hydrolysis of phosphoramidite building blocks 12 and 16 in 95%aqueous MeCN at 25° C. was followed by reverse-phase HPLC. It wasdetermined that, under the conditions used, the half-life of compounds12 and 16 was 48 and 33 h, respectively. This demonstrates thatsolutions of phosphoramidite reagents according to the present inventioncan be safely stored on an oligonucleotide synthesizer for 2-3 days,which is sufficient for most applications.

The use of novel phosphoramidite building blocks described herein insolid phase synthesis of oligonucleotides is illustrated with respect tocompound 12:

d(Tp Tp Tp Tp TT TTT T)

wherein internucleosidic phosphate linkages p are phosphotriesterlinkages with 1-[2-[[2-(β-D-glucopyranosyloxy)ethyl]oxy]ethyl]modification (SEQ ID NO:6).

Oligonucleotides 61-64 were synthesized by the phosphoramidite method.Briefly, a commercial solid support, DMT-T CPG1000 (1.0 μmol) was placedin a synthetic column and installed on ABI394 synthesizer. The standardsynthetic cycle useful in assemblying oligonucleotides with phosphatebackbone comprised the following steps:

-   -   (a) detritylation of the solid phase-bound material with 3%        dichloroacetic acid in dichloromethane followed by washing with        acetonitrile;    -   (b) coupling of nucleoside phosphoramidite building block (0.1 M        in acetonitrile) required by the sequence to the solid        support-bound material in the presence of 1H-tetrazole (0.45 M        in acetonitrile);    -   (c) capping of unreacted solid support-bound hydroxy groups with        a mixture of acetic anhydride and N-methyl imidazole and    -   (d) oxidation of the solid support-bound phosphite triester        groups with iodine (0.05 M in a mixture of pyridine,        tetrahydrofurane, water).        Preparation of oligonucleotide phosphorothioates was conducted        as disclosed in U.S. Pat. No. 7,723,528. Accordingly, steps (a)        and (b) remained as disclosed above. The iodine solution was        replaced with a solution of        N′-(3-thioxo-3H-1,2,4-dithiazol-5-yl)-N,N-dimethylmethanimidamide        (0.025 M in pyridine). Upon completion of coupling step (b) and        washing the solid phase with acetonitrile, the sulfurization        step was conducted. The capping step followed the sulfurization.        The cycle appropriate for the assembly of the desired        oligonucleotide was repeated as required by the sequence in        preparation.

The synthetic cycle for incorporation of the phosphoramidite buildingblocks described herein was different from those disclosed above in thatthe coupling time for the incorporation of all novel phosphoramiditebuilding blocks was extended to 1 min.

Following the general procedures disclosed above, the parent, unmodifiedoligonucleotide 60, plus oligonucleotides 61-64 wherein Tp denotes theresidues of thymidine-3′-[[(β-D-glucopyranosyl)oxy]propyl]phosphate 65,were assembled:

(SEQ ID NO: 1) 60: TTT TTT TTT T, parent natural oligonucleotide,(SEQ ID NO: 2) 61: Tp TTT TTT TTT, (SEQ ID NO: 3) 62: (Tp)₂ TTT TTT TT,(SEQ ID NO: 4) 63: (Tp)₃ TTT TTT T, and (SEQ ID NO: 5)64: (Tp)₄ TTT TTT.

To determine the optimal deprotection conditions, solid support-boundoligonucleotides 61-64 thus obtained were treated under the followingconditions suitable for removing all phosphate- and base-protectinggroups from synthetic oligonucleotides:

-   -   A. Concentrated ammonium hydroxide, room temperature, 18 h;    -   B. Concentrated aqueous ammonium hydroxide, 65° C., 8 h;    -   C. 0.05 M K₂CO₃ in anhydrous MeOH, room temperature, 4 h;    -   D. 0.5 M Piperidine in anhydrous acetonitrile, room temperature,        15 min, followed by a mixture of 1,2-diaminoethane and toluene        (50:50 v/v), room temperature, 4 h;    -   E. 25% Aqueous t-butylamine, 65° C., 4 h;    -   F. Mixture of concentrated aqueous ammonium hydroxide and 40%        aqueous methylamine (50:50 v/v), room temperature, 120 min.

Upon completion of the deprotection under the conditions A, B, E, and F,the liquid phase was collected and evaporated in vacuo to dryness. Theresidue was dissolved in water (1 mL) and analyzed by reverse-phase HPLCand by ES MS.

Upon completion of the deprotection under the condition C, the reactionmixtures were neutralized by addition of 0.25 M aqueous NaH₂PO₄ (250 μLper 1 mL of deprotection solution). The solid phase was spun down; theliquid phase was collected and analyzed as described above.

Upon completion of the deprotection under the condition D, the solidphase was spun down, and the liquid phase was discarded. The solid phasewas washed with toluene (2×1 mL) and with acetonitrile (2×1 mL). Thesolid phase was suspended in water (1 mL) and the suspension wascentrifugated. The liquid phase was collected and analyzed as describedabove.

The results of HPLC and LC-ES MS analysis showed that deprotection underconditions A, C, D, and F resulted in products containing over 95% ofthe desired oligonucleotides. In contrast, the use of the conditions Band E led to the formation of the parent oligonucleotide 60 (SEQ IDNO: 1) where the (glucopyranosyloxy)propyl modifier was removed from thephosphotriester moiety. A more detailed analysis of the crudeoligonucleotide products showed that conditions A and C were the mostappropriate for deprotection of phosphotriester-derivatizedoligonucleotides.

In the next round of experiments, oligonucleotides 67-72 were assembledand deprotected under conditions A and C as described above:

-   -   67: Tp Tp Tp Tp TT TTT T (SEQ ID NO: 6) wherein nucleoside        phosphate moieties Tp are phosphotriester moieties 75 (FIG. 9)        with 1-[2-[[2-(β-D-glucopyranosyloxy)ethyl]oxy]ethyl]        modification;    -   68: Tp Tp Tp Tp T Tp Tp Tp Tp T (SEQ ID NO: 7) wherein        nucleoside phosphate moieties Tp are phosphotriester moieties 65        (FIG. 9) with 1-[3-(β-D-glucopyranosyloxy)propyl]modification;    -   69: Tp Tp Tp Tp Tp Tp Tp Tp Tp T (SEQ ID NO: 8) wherein all        nucleoside phosphate moieties Tp are phosphotriester moieties 65        (FIG. 9) with 1-[3-(β-D-glucopyranosyloxy)propyl] modification;    -   70: Tp Tp Tp Tp Tp Tp Tp Tp Tp T (SEQ ID NO: 9) wherein all        nucleoside phosphate moieties Tp are phosphotriester moieties 75        (FIG. 9) with 1-[2-[[2-(β-D-glucopyranosyloxy)ethyl]oxy]ethyl]        modification;    -   71: Tp Tp Tp Tp TT TTT T (SEQ ID NO: 10) wherein nucleoside        phosphate moieties Tp are phosphotriester moieties 79 (FIG. 9)        with 1-[2-[[2-(N-acetyl-2-deoxy-2-amino-β-D-galactopyranosyloxy)        ethyl]oxy]ethyl] modification; and    -   72: Tp Tp Tp Tp T Tp Tp Tp Tp T (SEQ ID NO: 11) wherein        nucleoside phosphate moieties Tp are phosphotriester moieties 79        (FIG. 9) with        1-[2-[[2-(N-acetyl-2-deoxy-2-amino-β-D-galactopyranosyloxy)        ethyl]oxy]ethyl] modification.

Analysis of crude oligonucleotide products by reverse-phase HPLC andLC-ES MS confirmed that both deprotection conditions A and C wereappropriate for preparation of highly-modified 10-mer oligonucleotides68, 69, 70, and 71 comprising 8 to 9 phosphotriester moieties, thusconfirming the stability of the novel modification of internucleosidicphosphate groups disclosed in the present invention. Further comparisonof HPLC profiles obtained for oligonucleotides 64, 67, and 71 revealedno appreciable difference in hydrolytic stability between nucleosidephosphate residues 65, 75, and 79 under the basic deprotectionconditions.

Finally, full-length oligonucleotide phosphorothioates 81-84 weresynthesized and deprotected under the condition A:

-   -   81: d(TGT GAG TAC CAC TGA TTC) (SEQ ID NO: 12) phosphorothioate        wherein all internucleoside moieties are phosphorothioate        triester moieties with 1-[3-(β-D-glucopyranosyloxy)propyl]        modification as exemplified for the triester of        thymidine-3′-monophosphorothioate by structure 66 (FIG. 9);    -   82: d(TpGpTp GAG TAC CAC TGAp TpTpA) (SEQ ID NO: 13)        phosphorothioate wherein all internucleoside moieties are        phosphorothioate moieties and Tp and Ap are phosphorothioate        triester moieties with        1-[3-(N-acetyl-2-deoxy-2-amino-β-D-glucopyranosyloxy)propyl]        modification as exemplified for the triester of        thymidine-3′-monophosphorothioate by structure 78 (FIG. 9);    -   83: d(TpGpTp GAG TAC CAC TGAp TpTpA) phosphorothioate (SEQ ID        NO: 14) wherein all internucleoside moieties are        phosphorothioate moieties and Tp and Ap are phosphorothioate        triester moieties with        1-[2-[[2-(N-acetyl-2-deoxy-2-amino-β-D-galactopyranosyloxy)ethyl]oxy]ethyl]        modification as exemplified for the triester of        thymidine-3′-monophosphorothioate by structure 80 (FIG. 9);    -   84: dTp dGp dTp GAG UAC CAC UG dAp dTp dTp dA phosphorothioate        (SEQ ID NO: 15) wherein all internucleoside moieties are        phosphorothioate moieties and Tp and Ap are phosphorothioate        triester moieties with        1-[2-[[2-(N-acetyl-2-deoxy-2-amino-β-D-galactopyranosyloxy)ethyl]oxy]ethyl]        modification as exemplified for the triester of        thymidine-3′-monophosphorothioate by structure 80 (FIG. 9).

Oligonucleotide 81 was characterized by reverse-phase HPLC, andoligonucleotides 82-84 were characterized by reverse-phase HPLC, ionexchange HPLC, and LC-MS with electron-spray ionization. Importantly,oligonucleotides 68 and 72 bearing only one negative charge and 69, 70,and 81 wherein all negatively charged internucleosidic phosphates werereplaced by neutral phosphotriested moieties were readily soluble inaqueous media.

Accordingly, the efficient preparation of monosaccharide-derivatizedphosphotriester analogs of oligonucleotides and their phosphorothioateanalogs using the novel phosphoramidite building blocks described hereinhas been demonstrated. Said phosphoramidite building blocks can bereadily synthesized by artisans possessing ordinary skills.Conveniently, phosphotriester oligonucleotide analogs synthesized usingsaid phosphoramidite building blocks are stable under the basicdeprotection conditions as opposed to methyl triester analogs (U.S. Pat.No. 5,955,599), O-acyloxyaryl triester analogs (Iyer, R. P.; et al.Bioorg. Med. Chem. Lett. 1997, 7 (7), 871-876), acylaminoethyl analogs(U.S. Pat. No. 6,121,437, U.S. Pat. No. 6,610,837, US 2001/0044529, WO2003/048179, and WO 2006/065751), (pivaloylthio)ethyl triester andsimilar analogs (U.S. Pat. No. 6,919,437, WO2010/039543 and inWO2014/031575).

EXAMPLES

The following examples are intended to further illustrate certainpreferred embodiments of the invention and are not intended to belimiting in their nature.

General Information

Protected 2′-deoxynucleoside 2-cyanoethyl phosphoramidites, protectedribonucleoside 2-cyanoethyl phosphoramidites, and all ancillary reagentsfor oligonucleotide synthesis were purchased from Glen Research(Sterling, Va.). Sulfurizing reagent,N′-(3-thioxo-3H-1,2,4-dithiazol-5-yl)-N,N-dimethylmethanimidamide wasprepared as disclosed in U.S. Pat. No. 7,723,582. Anhydrous MeCN waspurchased from Honeywell Burdick & Jackson (Muskegon, Mich.). Protected2′-deoxynucleosides, 2′-O-methylribonucleosides, and2′-fluoro-2′-deoxyribonucleosides were purchased from Rasayan, Inc.(Encinitas, Calif.). Tri-tetra- and pentaethyleneglycols were purchasedfrom Sigma-Aldrich (Milwaukee, Wis.). All other chemicals were purchasedfrom TCI America (Portland, Oreg.).

Example 15′-O-(4,4′-Dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[3-[(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)oxy]propyl]phosphino]thymidine(12)

Solution of bis(N,N-diisopropylamino) chlorophosphite (2.94 g, 11 mmol)in anhydrous dichloromethane (10 mL) was added dropwise to a stirredsolution of 5′-O-(4,4′-dimethoxytrityl)thymidine, 1, (5.88 g, 10.8 mmol)and N-ethyl-N,N-diisopropylamine (1.44 g, 11.1 mmol) in anhydrousdichloromethane (25 mL) at −20° C. The solution was allowed to stir atroom temperature for 2 h. To the obtained mixture, 3-hydroxypropyl2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside (4.47 g, 11 mmol, prepared asdisclosed in Karjala, S. and Link, K. P., J. Amer. Chem. Soc., 1940, 62,917-922) and 1H-tetrazole (0.45 M in MeCN, 7.8 mL) were added followedby stirring at room temperature for 14 h. The reaction mixture wasquenched by addition of excess 5% aqueous NaHCO₃. The emulsion wasdiluted with 5% aqueous NaHCO₃ (200 mL), and the product was extractedwith ethyl acetate (3×100 mL). Extracts were washed with brine, driedover anhydrous Na₂SO₄, and evaporated to oil. The product was isolatedchromatographically on a silica gel column eluted with a step gradientof ethyl acetate (from 20 to 80%) plus triethylamine (5%) in hexane.Collected fractions were evaporated to give the title compound as amixture of S_(p) and R_(p) diastereomers (8.11 g, 69.5%). ³¹P NMR(CD₃CN): δ 147.94, 147.74. ¹H NMR (CD₃CN): δ 8.941 (1H, br. s);7.467-7.431 (3H, m); 7.331-7.291 (6H, m); 7.28-7.20 (1H, m); 6.886-6.855(4H, m) 6.250-6.211 (1H, m); 5.247-5.171 (1H, m); 5.016-4.977 (1H, m);4.866-4.822 (1H, m); 4.600-4.526 (1H, m); 4.203-4.168 (1H, m);4.054-4.038 (2H, m); 3.90-3.83 (0.5H, m); 3.83-3.70 (2.5H, m); 3.765(3H, s); 3.761 (3H, s); 3.68-3.45 (5H, m); 3.329-3.273 (2H, m);2.45-2.38 (0.5H, m); 2.37-2.28 (1.5H, m); 2.015-1.929 (12H, m);1.82-1.74 (1H, m); 1.72-1.64 (1H, m); 1.498 (1.5H, d, J=1.0 Hz); 1.476(1.5H, d, J=1.0 Hz); 1.155-1.117 (9H, m); 1.033 (3H, d, J=6.8 Hz).

Example 2N⁴-Acetyl-5′-O-(4,4′-dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[3-[(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)oxy]propyl]phosphino]-2′-deoxycytidine(13a)

Solution of bis(N,N-diisopropylamino) chlorophosphite (5.27 g, 19.75mmol) in anhydrous dichloromethane (20 mL) was added dropwise to astirred solution ofN⁴-benzoyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxycytidine, 9a, (10.01 g,15.8 mmol, Rasayan, Inc., Encinitas, Calif.) andN-ethyl-N,N-diisopropylamine (3.83 g, 29.6 mmol) in anhydrousdichloromethane (30 mL) at −20° C. The solution was allowed to stir atroom temperature for 2 h. To the obtained mixture, 3-hydroxypropyl2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside, 1, (7.06 g, 17.38 mmol) and1H-tetrazole (0.45 M in MeCN, 10.0 mL, 4.5 mmol) were added followed bystirring at room temperature for 14 h. The reaction mixture was quenchedby addition of excess 5% aqueous NaHCO₃. The emulsion was diluted with5% aqueous NaHCO₃ (100 mL), and the product was extracted with ethylacetate (3×100 mL). Extracts were washed with brine, dried overanhydrous Na₂SO₄, and evaporated to oil. The product was isolatedchromatographically on a silica gel column eluted with a step gradientfrom ethyl acetate—TEA—hexane (10:3:87) to ethyl acetate—TEA (94:6).Collected fractions were evaporated to give the title compound (13.30 g,72.0%). ³¹P NMR (mixture of diastereomers, CD₃CN): δ 147.86, 147.53.

Example 3N⁴-Phenoxyacetyl-5′-O-(4,4′-dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[3-[(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)oxy]propoxy]phosphinyl]-2′-deoxycytidine(13b)

Solution of bis(N,N-diisopropylamino) chlorophosphite (4.75 g, 17.81mmol) in anhydrous dichloromethane (20 mL) was added dropwise to astirred solution ofN⁴-phenoxyacetyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxycytidine, 9b, (9.46g, 14.25 mmol, Rasayan, Inc., Encinitas, Calif.) andN-ethyl-N,N-diisopropylamine (3.45 g, 26.7 mmol) in anhydrousdichloromethane (30 mL) at −20° C. The solution was allowed to stir atroom temperature for 2 h. To the obtained mixture, 3-hydroxypropyl2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside, 1, (6.37 g, 15.68 mmol) and1H-tetrazole (0.45 M in MeCN, 12.7 mL) were added followed by stirringat room temperature for 14 h. The reaction mixture was quenched byaddition of excess 5% aqueous NaHCO₃. The emulsion was diluted with 5%aqueous NaHCO₃ (100 mL), and the product was extracted with ethylacetate (3×100 mL). Extracts were washed with brine, dried overanhydrous Na₂SO₄, and evaporated to oil. The product was isolatedchromatographically on a silica gel column eluted with a step gradientfrom ethyl acetate—TEA—hexane (10:2:87) to TEA—ethyl acetate (10:90).Collected fractions were evaporated to give the title compound (11.60 g,67.9%). ³¹P NMR (mixture of diastereomers, CD₃CN): δ 148.12, 147.77.

Example 4N⁴-Acetyl-5′-O-(4,4′-dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[3-[(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)oxy]propoxy]phosphinyl]-2′-deoxycytidine(13c)

Solution of bis(N,N-diisopropylamino) chlorophosphite (4.62 g, 17.32mmol) in anhydrous dichloromethane (15 mL) was added dropwise to astirred solution ofN⁴-acetyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxycytidine, 9c, (7.83 g,13.69 mmol, Rasayan, Inc., Encinitas, Calif.) andN-ethyl-N,N-diisopropylamine (2.12 g, 16.42 mmol) in anhydrousdichloromethane (30 mL) at −20° C. The solution was allowed to stir atroom temperature for 2 h. To the obtained mixture, 3-hydroxypropyl2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside, 1, (6.40 g, 15.75 mmol) and1H-tetrazole (0.45 M in MeCN, 10.0 mL, 4.5 mmol) were added followed bystirring at room temperature for 14 h. The reaction mixture was quenchedby addition of excess 5% aqueous NaHCO₃. The emulsion was diluted with5% aqueous NaHCO₃ (100 mL), and the product was extracted with ethylacetate (3×100 mL). Extracts were washed with brine, dried overanhydrous Na₂SO₄, and evaporated to oil. The product was isolatedchromatographically on a silica gel column eluted with a step gradientfrom ethyl acetate—TEA—hexane (10:2:87) to TEA—ethyl acetate (6:94).Collected fractions were evaporated to give the title compound (6.78 g,44.7%). ³¹P NMR (mixture of diastereomers, CD₃CN): δ 147.97, 147.63. ¹HNMR (fast diastereomer, CD₃CN): δ 8.805 (1H, br. s); 8.172 (1H, d, J=7.5Hz); 7.429-7.412 (2H, m); 7.322-7.280 (6H, m); 7.257-7.228 (1H, m);7.040 (1H, d, J=7.5 Hz); 6.878-6.848 (4H, m); 6.087 (1H, dd, J=6.5 Hz,4.8 Hz); 5.323 (1H, t, J=9.6 Hz); 4.997 (1H, t, J=9.8 Hz); 4.838 (1H,dd, J=9.8, 8.0 Hz); 4.627 (1H, d, J=8.1 Hz); 4.50-4.48 (1H, m); 4.230(1H, dd, J=12.3, 5.0 Hz); 4.103 (1H, m); 4.042 (1H, dd, J=12.3, 2.4 Hz);3.885-3.849 (2H, m); 3.768 (6H, s); 3.625-3.510 (5H, m); 3.41-3.32 (2H,m); 2.573 (1H, d, J=13.7 Hz, t, J=6.7 Hz); 2.261 (1H, d, J=13.7 Hz, d,J=6.5 Hz, d, J=4.8 Hz); 2.116 (3H, s); 2.018 (3H, s); 1.975 (3H, s);1.956-1.784 (6H, m); 1.772 (2H, tt, J=6.2 Hz); 1.123 (3H, d, J=6.8 Hz);1.046 (3H, d, J=6.8 Hz).

Example 5N⁶-Benzoyl-5′-O-(4,4′-dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[3-[(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)oxy]propoxy]phosphinyl]-2′-deoxyadenosine(14a)

Solution of bis(N,N-diisopropylamino) chlorophosphite (4.12 g, 15.44mmol) in anhydrous dichloromethane (15 mL) was added dropwise to astirred solution ofN⁶-benzoyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyadenosine, 10a, (8.12 g,12.35 mmol, Rasayan, Inc., Encinitas, Calif.) andN-ethyl-N,N-diisopropylamine (1.99 g, 15.44 mmol) in anhydrousdichloromethane (30 mL) at −20° C. The solution was allowed to stir atroom temperature for 2 h. To the obtained mixture, 3-hydroxypropyl2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside, 1, (6.07 g, 14.94 mmol) and1H-tetrazole (0.45 M in MeCN, 8.2 mL) were added followed by stirring atroom temperature for 14 h. The reaction mixture was quenched by additionof excess 5% aqueous NaHCO₃. The emulsion was diluted with 5% aqueousNaHCO₃ (100 mL), and the product was extracted with ethyl acetate (3×100mL). Extracts were washed with brine, dried over anhydrous Na₂SO₄, andevaporated to oil. The product was isolated chromatographically on asilica gel column eluted with a step gradient from ethylacetate—TEA—hexane (10:2:87) to TEA—ethyl acetate (5:95). Collectedfractions were evaporated to give the title compound (11.49 g, 78.0%).³¹P NMR (mixture of diastereomers, CD₃CN): δ 147.87, 147.58. ¹H NMR(mixture of diastereomers, CD₃CN): δ 9.318 (1H, br. s); 8.591 (1H, br.d, J=3.3 Hz); 8.263 (1H, s); 8.997 (1H, br d, J=7.2 Hz); 7.646 (1H, t,J=7.3 Hz); 7.551 (2H, t, J=7.7 Hz); 7.39-7.34 (2H, m); 7.25-7.16 (7H,m); 6.80-6.74 (4H, m); 6.48-6.42 (1H, m); 5.24-5.16 (1H, m); 5.05-4.96(1H, m); 4.90-4.78 (2H, m); 4.556 (1H, dd, J=8.0, 5.1 Hz); 4.26-4.17(2H, m); 4.08-3.98 (1.5H, m); 3.91-3.78 (1.5H, m); 3.740, 3.736, 3.733,3.728 (total 6H, singlets); 3.78-3.69 (1H, m); 3.64-3.50 (5H, m);3.48-3.24 (2H, m); 3.060 (1H, sept, J=6.4 Hz); 2.68-2.52 (1H, m);2.00-1.90 (12H, m); 1.795 (1H, pent., J=6.1 Hz); 1.711 (1H, pent., J=6.1Hz); 1.20-1.12 (9H, m); 1.080 (3H, d, J=6.8 Hz).

Example 6N⁶-Phenoxyacetyl-5′-O-(4,4′-dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[3-[(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)oxy]propoxy]phosphinyl]-2′-deoxyadenosine(14b)

Solution of bis(N,N-diisopropylamino) chlorophosphite (3.50 g, 13.13mmol) in anhydrous dichloromethane (15 mL) was added dropwise to astirred solution ofN⁶-phenoxyacetyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyadenosine, 10b,(7.22 g, 10.50 mmol, Rasayan, Inc., Encinitas, Calif.) andN-ethyl-N,N-diisopropylamine (2.55 g, 19.74 mmol) in anhydrousdichloromethane (30 mL) at −20° C. The solution was allowed to stir atroom temperature for 2 h. To the obtained mixture, 3-hydroxypropyl2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside, 1, (4.69 g, 11.55 mmol) and1H-tetrazole (0.45 M in MeCN, 9.3 mL) were added followed by stirring atroom temperature for 14 h. The reaction mixture was quenched by additionof excess 5% aqueous NaHCO₃. The emulsion was diluted with 5% aqueousNaHCO₃ (100 mL), and the product was extracted with ethyl acetate (3×100mL). Extracts were washed with brine, dried over anhydrous Na₂SO₄, andevaporated to oil. The product was isolated chromatographically on asilica gel column eluted with a step gradient from ethylacetate—TEA—hexane (10:2:87) to TEA—ethyl acetate (10:90). Collectedfractions were evaporated to give the title compound (9.05 g, 70.5%).³¹P NMR (mixture of diastereomers, CD₃CN): δ 148.54, 148.01.

Example 7N⁶-(4-i-Propylphenoxy)acetyl-5′-O-(4,4′-dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[3-[(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)oxy]propoxy]phosphinyl]-2′-deoxyadenosine(14c)

Solution of bis(N,N-diisopropylamino) chlorophosphite (3.94 g, 14.75mmol) in anhydrous dichloromethane (15 mL) was added dropwise to astirred solution of N⁶-(4-i-propylphenoxy)acetyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyadenosine, 10c, (8.61 g,11.80 mmol, Rasayan, Inc., Encinitas, Calif.) andN-ethyl-N,N-diisopropylamine (2.87 g, 22.18 mmol) in anhydrousdichloromethane (30 mL) at −20° C. The solution was allowed to stir atroom temperature for 2 h. To the obtained mixture, 3-hydroxypropyl2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside, 1, (5.27 g, 12.98 mmol) and1H-tetrazole (0.45 M in MeCN, 10.5 mL) were added followed by stirringat room temperature for 14 h. The reaction mixture was quenched byaddition of excess 5% aqueous NaHCO₃. The emulsion was diluted with 5%aqueous NaHCO₃ (100 mL), and the product was extracted with ethylacetate (3×100 mL). Extracts were washed with brine, dried overanhydrous Na₂SO₄, and evaporated to oil. The product was isolatedchromatographically on a silica gel column eluted with a step gradientfrom ethyl acetate—TEA—hexane (10:2:87) to TEA—ethyl acetate (10:90).Collected fractions were evaporated to give the title compound (10.44 g,69.9%). ³¹P NMR (mixture of diastereomers, CD₃CN): δ 148.22, 147.72.

Example 8N⁶—(N,N-Dimethylformamidino)-5′-O-(4,4′-dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[3-[(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)oxy]propoxy]phosphinyl]-2′-deoxyadenosine(14d)

Solution of bis(N,N-diisopropylamino) chlorophosphite (4.67 g, 17.5mmol) in anhydrous dichloromethane (15 mL) was added dropwise to astirred solution ofN⁶—(N,N-dimethylformamidino)-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyadenosine,10d, (8.52 g, 14.0 mmol, Rasayan, Inc., Encinitas, Calif.) andN-ethyl-N,N-diisopropylamine (3.40 g, 26.32 mmol) in anhydrousdichloromethane (30 mL) at −20° C. The solution was allowed to stir atroom temperature for 2 h. To the obtained mixture, 3-hydroxypropyl2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside, 1, (6.25 g, 15.4 mmol) and1H-tetrazole (0.45 M in MeCN, 12.44 mL) were added followed by stirringat room temperature for 14 h. The reaction mixture was quenched byaddition of excess 5% aqueous NaHCO₃. The emulsion was diluted with 5%aqueous NaHCO₃ (100 mL), and the product was extracted with ethylacetate (3×100 mL). Extracts were washed with brine, dried overanhydrous Na₂SO₄, and evaporated to oil. The product was isolatedchromatographically on a silica gel column eluted with a step gradientfrom ethyl acetate—TEA—hexane (10:2:87) to TEA—ethyl acetate (10:90).Collected fractions were evaporated to give the title compound (12.56 g,78.4%). ³¹P NMR (mixture of diastereomers, CD₃CN): δ 148.65, 148.12.

Example 9N²-Isobutyryl-5′-O-(4,4′-dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[3-[(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)oxy]propoxy]phosphinyl]-2′-deoxyguanosine(15a)

Solution of bis(N,N-diisopropylamino) chlorophosphite (4.27 g, 16.0mmol) in anhydrous dichloromethane (15 mL) was added dropwise to astirred solution ofN²-isobutyryl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyguanosine, 11a, (8.53g, 13.33 mmol, Rasayan, Inc., Encinitas, Calif.) andN-ethyl-N,N-diisopropylamine (2.15 g, 16.66 mmol) in anhydrousdichloromethane (30 mL) at −20° C. The solution was allowed to stir atroom temperature for 2 h. To the obtained mixture, 3-hydroxypropyl2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside, 1, (6.50 g, 16.0 mmol) and1H-tetrazole (0.45 M in MeCN, 10.0 mL, 4.5 mmol) were added followed bystirring at room temperature for 14 h. The reaction mixture was quenchedby addition of excess 5% aqueous NaHCO₃. The emulsion was diluted with5% aqueous NaHCO₃ (100 mL), and the product was extracted with ethylacetate (3×100 mL). Extracts were washed with brine, dried overanhydrous Na₂SO₄, and evaporated to oil. The product was isolatedchromatographically on a silica gel column eluted with a step gradientfrom ethyl acetate—TEA—hexane (20:5:75) to TEA—ethyl acetate (5:95).Collected fractions were evaporated to give the title compound as anoff-white foam (8.54 g, 54.5%). ³¹P NMR (mixture of diastereomers,CD₃CN): δ 147.96, 147.62. ¹H NMR (mixture of diastereomers, CD₃CN): δ7.817, 7.812 (total 1H, s, s); 7.43-7.36 (2H, m); 7.29-7.18 (7H, m);6.83-6.72 (4H, m); 6.249-6.214 (1H, m); 5.24-5.14 (1H, m); 5.03-4.94(1H, m); 4.89-4.81 (1H, m); 4.64-4.52 (2H, m); 4.22-4.12 (2H, m);4.07-3.98 (1H, m); 3.90-3.88 (1H, m); 3.748, 3.744, 3.740 (total 6H, s,s, s); 3.77-3.69 (1H, m); 3.68-3.49 (5H, m); 3.37-3.28 (1H, m);3.27-3.19 (1H, m); 2.94-2.83 (1H, m); 2.601 (1H, sept., J=6.8 Hz);2.58-2.43 (1H, m); 1.99-1.93 (12H, m); 1.777 (1.5H, pent., J=6.2 Hz);1.698 (0.5H, m); 1.186-1.131 (12H, m); 1.063 (3H, d, J=6.8 Hz).

Example 10N²-Phenoxyacetyl-5′-O-(4,4′-dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[3-[(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)oxy]propoxy]phosphinyl]-2′-deoxyguanosine(15b)

Solution of bis(N,N-diisopropylamino) chlorophosphite (2.83 g, 10.6mmol) in anhydrous dichloromethane (10 mL) was added dropwise to astirred solution ofN²-phenoxyacetyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyguanosine, 11b,(5.98 g, 8.5 mmol, Rasayan, Inc., Encinitas, Calif.) andN-ethyl-N,N-diisopropylamine (2.06 g, 16.0 mmol) in anhydrousdichloromethane (25 mL) at −20° C. The solution was allowed to stir atroom temperature for 2 h. To the obtained mixture, 3-hydroxypropyl2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside, 1, (3.97 g, 9.78 mmol) and1H-tetrazole (0.45 M in MeCN, 7.6 mL) were added followed by stirring atroom temperature for 14 h. The reaction mixture was quenched by additionof excess 5% aqueous NaHCO₃. The emulsion was diluted with 5% aqueousNaHCO₃ (100 mL), and the product was extracted with ethyl acetate (3×100mL). Extracts were washed with brine, dried over anhydrous Na₂SO₄, andevaporated to oil. The product was isolated chromatographically on asilica gel column eluted with a step gradient from ethylacetate—TEA—hexane (20:5:75) to TEA—ethyl acetate (10:90). Collectedfractions were evaporated to give the title compound as an off-whitefoam (6.54 g, 62.1%). ³¹P NMR (mixture of diastereomers, CD₃CN): δ148.90, 148.48.

Example 11N²-(4-i-Propylphenoxy)acetyl-5′-O-(4,4′-dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[3-[(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)oxy]propoxy]phosphinyl]-2′-deoxyguanosine(15c)

Solution of bis(N,N-diisopropylamino) chlorophosphite (3.33 g, 12.5mmol) in anhydrous dichloromethane (10 mL) was added dropwise to astirred solution ofN²-(4-i-propylphenoxy)acetyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyguanosine,11c, (7.46 g, 10.0 mmol, Rasayan, Inc., Encinitas, Calif.) andN-ethyl-N,N-diisopropylamine (2.43 g, 18.8 mmol) in anhydrousdichloromethane (25 mL) at −20° C. The solution was allowed to stir atroom temperature for 2 h. To the obtained mixture, 3-hydroxypropyl2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside, 1, (4.67 g, 11.5 mmol) and1H-tetrazole (0.45 M in MeCN, 8.9 mL) were added followed by stirring atroom temperature for 14 h. The reaction mixture was quenched by additionof excess 5% aqueous NaHCO₃. The emulsion was diluted with 5% aqueousNaHCO₃ (50 mL), and the product was extracted with ethyl acetate (3×100mL). Extracts were washed with brine, dried over anhydrous Na₂SO₄, andevaporated to oil. The product was isolated chromatographically on asilica gel column eluted with a step gradient from ethylacetate—TEA—hexane (20:5:75) to TEA—ethyl acetate (10:90). Collectedfractions were evaporated to give the title compound as an off-whitefoam (8.10 g, 63.3%). ³¹P NMR (mixture of diastereomers, CD₃CN): δ148.88, 148.32.

Example 12N²—(N,N-Dimethylformamidino)-5′-O-(4,4′-dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[3-[(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)oxy]propoxy]phosphinyl]-2′-deoxyguanosine(15d)

Solution of bis(N,N-diisopropylamino) chlorophosphite (3.67 g, 13.75mmol) in anhydrous dichloromethane (10 mL) was added dropwise to astirred solution ofN²—(N,N-dimethylformamidino)-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyguanosine,11d, (6.87 g, 11.0 mmol, Rasayan, Inc., Encinitas, Calif.) andN-ethyl-N,N-diisopropylamine (2.67 g, 20.7 mmol) in anhydrousdichloromethane (25 mL) at −20° C. The solution was allowed to stir atroom temperature for 2 h. To the obtained mixture, 3-hydroxypropyl2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside, 1, (2.92 g, 6.7 mmol) and1H-tetrazole (0.45 M in MeCN, 9.8 mL) were added followed by stirring atroom temperature for 14 h. The reaction mixture was quenched by additionof excess 5% aqueous NaHCO₃. The emulsion was diluted with 5% aqueousNaHCO₃ (50 mL), and the product was extracted with ethyl acetate (3×100mL). Extracts were washed with brine, dried over anhydrous Na₂SO₄, andevaporated to oil. The product was isolated chromatographically on asilica gel column eluted with a step gradient from ethylacetate—TEA—hexane (20:5:75) to TEA—ethyl acetate (10:90). Collectedfractions were evaporated to give the title compound as an off-whitefoam (8.10 g, 63.3%). ³¹P NMR (mixture of diastereomers, CD₃CN): δ148.88, 148.32.

Example 135′-O-(4,4′-Dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[2-[[2-[(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)oxy]ethyl]oxy]ethoxy]phosphinyl]thymidine (16)

Solution of bis(N,N-diisopropylamino) chlorophosphite (1.86 g, 6.98mmol) in anhydrous dichloromethane (5 mL) was added dropwise to astirred solution of 5′-O-(4,4′-dimethoxytrityl)thymidine, 8, (3.17 g,5.82 mmol) and N-ethyl-N,N-diisopropylamine (0.99 g, 7.68 mmol) inanhydrous dichloromethane (15 mL) at −20° C. The solution was allowed tostir at room temperature for 2 h. To the obtained mixture,2-[(2-hydroxyethyl)oxy]ethyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside,2, (2.92 g, 6.69 mmol, prepared as disclosed in Karjala, S. and Link, K.P., J. Amer. Chem. Soc., 1940, 62, 917-922) and 1H-tetrazole (0.45 M inMeCN, 3.9 mL, 1.76 mmol) were added followed by stirring at roomtemperature for 14 h. The reaction mixture was quenched by addition ofexcess 5% aqueous NaHCO₃. The emulsion was diluted with 5% aqueousNaHCO₃ (50 mL), and the product was extracted with ethyl acetate (3×100mL). Extracts were washed with brine, dried over anhydrous Na₂SO₄, andevaporated to oil. The product was isolated chromatographically on asilica gel column eluted with a step gradient from ethylacetate—TEA—hexane (20:2:77) to TEA—ethyl acetate (6:94). Collectedfractions were evaporated to give the title compound (4.87 g, 75.4%).³¹P NMR (mixture of diastereomers, CD₃CN): δ 148.23, 148.12. ¹H NMR(mixture of diastereomers, CD₃CN): δ 8.910 (1H, br. s); 7.48-7.40 (3H,m); 7.37-7.28 (6H, m); 7.28-7.20 (1H, m); 6.90-6.82 (4H, m); 6.28-6.20(1H, m); 5.26-5.18 (1H, m); 5.05-4.97 (1H, m); 4.89-4.82 (1H, m);4.68-4.53 (2H, m); 4.26-4.18 (1H, m); 4.12-4.01 (2H, m); 3.87-3.68 (8H,m, s, s); 3.68-3.41 (9H, m); 3.38-3.23 (2H, m); 2.45-2.29 (2H, m);2.02-1.92 (12H, m); 1.495 (1.5H, d, J=1.2 Hz); 1.471 (1.5H, d, J=1.2Hz); 1.17-1.11 (9H, m); 1.040 (3H, d, J=6.8 Hz).

Example 14N⁴-Benzoyl-5′-O-(4,4′-dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[2-[[2-[(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)oxy]ethyl]oxy]ethoxy]phosphinyl]-2′-deoxycytidine(17a)

Solution of bis(N,N-diisopropylamino) chlorophosphite (2.00 g, 7.5 mmol)in anhydrous dichloromethane (5 mL) was added dropwise to a stirredsolution of N⁴-benzoyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxycytidine, 9a,(3.80 g, 6.0 mmol) and N-ethyl-N,N-diisopropylamine (1.47 g, 11.4 mmol)in anhydrous dichloromethane (15 mL) at −20° C. The solution was allowedto stir at room temperature for 2 h. To the obtained mixture,2-[(2-hydroxyethyl)oxy]ethyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside,2, (3.01 g, 6.9 mmol) and 1H-tetrazole (0.45 M in MeCN, 5.3 mL) wereadded followed by stirring at room temperature for 14 h. The reactionmixture was quenched by addition of excess 5% aqueous NaHCO₃. Theemulsion was diluted with 5% aqueous NaHCO₃ (50 mL), and the product wasextracted with ethyl acetate (3×100 mL). Extracts were washed withbrine, dried over anhydrous Na₂SO₄, and evaporated to oil. The productwas isolated chromatographically on a silica gel column eluted with astep gradient from ethyl acetate—TEA—hexane (20:2:77) to TEA—ethylacetate (5:95). Collected fractions were evaporated to give the titlecompound (5.14 g, 71.4%). ³¹P NMR (mixture of diastereomers, CD₃CN): δ148.76, 148.39.

Example 15N⁴-Phenoxyacetyl-5′-O-(4,4′-dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[2-[[2-[(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)oxy]ethyl]oxy]ethoxy]phosphinyl]-2′-deoxycytidine(17b)

Solution of bis(N,N-diisopropylamino) chlorophosphite (2.00 g, 7.5 mmol)in anhydrous dichloromethane (5 mL) was added dropwise to a stirredsolution ofN⁴-phenoxyacetyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxycytidine, 9b, (3.98g, 6.0 mmol) and N-ethyl-N,N-diisopropylamine (1.47 g, 11.4 mmol) inanhydrous dichloromethane (15 mL) at −20° C. The solution was allowed tostir at room temperature for 2 h. To the obtained mixture,2-[(2-hydroxyethyl)oxy]ethyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside,2, (3.01 g, 6.9 mmol) and 1H-tetrazole (0.45 M in MeCN, 5.3 mL) wereadded followed by stirring at room temperature for 14 h. The reactionmixture was quenched by addition of excess 5% aqueous NaHCO₃. Theemulsion was diluted with 5% aqueous NaHCO₃ (50 mL), and the product wasextracted with ethyl acetate (3×100 mL). Extracts were washed withbrine, dried over anhydrous Na₂SO₄, and evaporated to oil. The productwas isolated chromatographically on a silica gel column eluted with astep gradient from ethyl acetate—TEA—hexane (20:2:77) to TEA—ethylacetate (5:95). Collected fractions were evaporated to give the titlecompound (4.86 g, 65.9%). ³¹P NMR (mixture of diastereomers, CD₃CN): δ148.05, 147.66.

Example 16N⁴-Acetyl-5′-O-(4,4′-dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[2-[[2-[(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)oxy]ethyl]oxy]ethoxy]phosphinyl]-2′-deoxycytidine (17c)

Solution of bis(N,N-diisopropylamino) chlorophosphite (2.00 g, 7.5 mmol)in anhydrous dichloromethane (5 mL) was added dropwise to a stirredsolution of N⁴-acetyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxycytidine, 9c,(3.43 g, 6.0 mmol) and N-ethyl-N,N-diisopropylamine (1.47 g, 11.4 mmol)in anhydrous dichloromethane (15 mL) at −20° C. The solution was allowedto stir at room temperature for 2 h. To the obtained mixture,2-[(2-hydroxyethyl)oxy]ethyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside,2, (3.01 g, 6.9 mmol) and 1H-tetrazole (0.45 M in MeCN, 5.3 mL) wereadded followed by stirring at room temperature for 14 h. The reactionmixture was quenched by addition of excess 5% aqueous NaHCO₃. Theemulsion was diluted with 5% aqueous NaHCO₃ (50 mL), and the product wasextracted with ethyl acetate (3×100 mL). Extracts were washed withbrine, dried over anhydrous Na₂SO₄, and evaporated to oil. The productwas isolated chromatographically on a silica gel column eluted with astep gradient from ethyl acetate—TEA—hexane (20:2:77) to TEA—ethylacetate (5:95). Collected fractions were evaporated to give the titlecompound (5.00 g, 73.3%). ³¹P NMR (mixture of diastereomers, CD₃CN): δ148.58, 148.14.

Example 17N⁶-Benzoyl-5′-O-(4,4′-dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[2-[[2-[(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)oxy]ethyl]oxy]ethoxy]phosphinyl]-2′-deoxyadenosine(18a)

Solution of bis(N,N-diisopropylamino) chlorophosphite (1.67 g, 6.25mmol) in anhydrous dichloromethane (5 mL) was added dropwise to astirred solution ofN⁶-benzoyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyadenosine, 10a, (3.29 g,5.0 mmol) and N-ethyl-N,N-diisopropylamine (1.23 g, 9.5 mmol) inanhydrous dichloromethane (15 mL) at −20° C. The solution was allowed tostir at room temperature for 2 h. To the obtained mixture,2-[(2-hydroxyethyl)oxy]ethyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside,2, (2.51 g, 5.75 mmol) and 1H-tetrazole (0.45 M in MeCN, 4.4 mL) wereadded followed by stirring at room temperature for 14 h. The reactionmixture was quenched by addition of excess 5% aqueous NaHCO₃. Theemulsion was diluted with 5% aqueous NaHCO₃ (50 mL), and the product wasextracted with ethyl acetate (3×100 mL). Extracts were washed withbrine, dried over anhydrous Na₂SO₄, and evaporated to oil. The productwas isolated chromatographically on a silica gel column eluted with astep gradient from ethyl acetate—TEA—hexane (20:2:77) to TEA—ethylacetate (5:95). Collected fractions were evaporated to give the titlecompound (4.33 g, 70.8%). ³¹P NMR (mixture of diastereomers, CD₃CN): δ148.32, 147.78.

Example 18N⁶-Phenoxyacetyl-5′-O-(4,4′-dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[2-[[2-[(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)oxy]ethyl]oxy]ethoxy]phosphinyl]-2′-deoxyadenosine (18b)

Solution of bis(N,N-diisopropylamino) chlorophosphite (1.67 g, 6.25mmol) in anhydrous dichloromethane (5 mL) was added dropwise to astirred solution ofN⁶-phenoxyacetyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyadenosine, 10b,(3.44 g, 5.0 mmol) and N-ethyl-N,N-diisopropylamine (1.23 g, 9.5 mmol)in anhydrous dichloromethane (15 mL) at −20° C. The solution was allowedto stir at room temperature for 2 h. To the obtained mixture,2-[(2-hydroxyethyl)oxy]ethyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside,2, (2.51 g, 5.75 mmol) and 1H-tetrazole (0.45 M in MeCN, 4.4 mL) wereadded followed by stirring at room temperature for 14 h. The reactionmixture was quenched by addition of excess 5% aqueous NaHCO₃. Theemulsion was diluted with 5% aqueous NaHCO₃ (50 mL), and the product wasextracted with ethyl acetate (3×100 mL). Extracts were washed withbrine, dried over anhydrous Na₂SO₄, and evaporated to oil. The productwas isolated chromatographically on a silica gel column eluted with astep gradient from ethyl acetate—TEA—hexane (20:2:77) to TEA—ethylacetate (5:95). Collected fractions were evaporated to give the titlecompound (3.98 g, 63.5%). ³¹P NMR (mixture of diastereomers, CD₃CN): δ148.44, 147.63.

Example 19N⁶-(4-i-Propylphenoxy)acetyl-5′-O-(4,4′-dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[2-[[2-[(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)oxy]ethyl]oxy] ethoxy]phosphinyl]-2′-deoxyadenosine (18c)

Solution of bis(N,N-diisopropylamino) chlorophosphite (1.67 g, 6.25mmol) in anhydrous dichloromethane (5 mL) was added dropwise to astirred solution of N⁶-(4-i-propylphenoxy)acetyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyadenosine, 10c, (3.65 g, 5.0mmol) and N-ethyl-N,N-diisopropylamine (1.23 g, 9.5 mmol) in anhydrousdichloromethane (15 mL) at −20° C. The solution was allowed to stir atroom temperature for 2 h. To the obtained mixture,2-[(2-hydroxyethyl)oxy]ethyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside,2, (2.51 g, 5.75 mmol) and 1H-tetrazole (0.45 M in MeCN, 4.4 mL) wereadded followed by stirring at room temperature for 14 h. The reactionmixture was quenched by addition of excess 5% aqueous NaHCO₃. Theemulsion was diluted with 5% aqueous NaHCO₃ (50 mL), and the product wasextracted with ethyl acetate (3×100 mL). Extracts were washed withbrine, dried over anhydrous Na₂SO₄, and evaporated to oil. The productwas isolated chromatographically on a silica gel column eluted with astep gradient from ethyl acetate—TEA—hexane (20:2:77) to TEA—ethylacetate (5:95). Collected fractions were evaporated to give the titlecompound (4.22 g, 65.1%). ³¹P NMR (mixture of diastereomers, CD₃CN): δ148.20, 147.69.

Example 20N⁶—(N,N-Dimethylformamidino)-5′-O-(4,4′-dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[2-[[2-[(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)oxy]ethyl]oxy] ethoxy]phosphinyl]-2′-deoxyadenosine (18d)

Solution of bis(N,N-diisopropylamino) chlorophosphite (1.67 g, 6.25mmol) in anhydrous dichloromethane (5 mL) was added dropwise to astirred solution ofN⁶—(N,N-dimethylformamidino)-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyadenosine,10d, (3.04 g, 5.0 mmol) and N-ethyl-N,N-diisopropylamine (1.23 g, 9.5mmol) in anhydrous dichloromethane (15 mL) at −20° C. The solution wasallowed to stir at room temperature for 2 h. To the obtained mixture,2-[(2-hydroxyethyl)oxy]ethyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside,2, (2.51 g, 5.75 mmol) and 1H-tetrazole (0.45 M in MeCN, 4.4 mL) wereadded followed by stirring at room temperature for 14 h. The reactionmixture was quenched by addition of excess 5% aqueous NaHCO₃. Theemulsion was diluted with 5% aqueous NaHCO₃ (50 mL), and the product wasextracted with ethyl acetate (3×100 mL). Extracts were washed withbrine, dried over anhydrous Na₂SO₄, and evaporated to oil. The productwas isolated chromatographically on a silica gel column eluted with astep gradient from ethyl acetate—TEA—hexane (20:2:77) to TEA—ethylacetate (5:95). Collected fractions were evaporated to give the titlecompound (4.56 g, 77.7%). ³¹P NMR (mixture of diastereomers, CD₃CN): δ148.88, 148.27.

Example 21N²-Isobutyryl-5′-O-(4,4′-dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[2-[[2-[(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)oxy]ethyl]oxy]ethoxy]phosphinyl]-2′-deoxyguanosine (19a)

Solution of bis(N,N-diisopropylamino) chlorophosphite (2.23 g, 8.4 mmol)in anhydrous dichloromethane (15 mL) was added dropwise to a stirredsolution of N²-isobutyryl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyguanosine,11a, (4.29 g, 6.7 mmol, Rasayan, Inc., Encinitas, Calif.) andN-ethyl-N,N-diisopropylamine (1.64 g, 12.7 mmol) in anhydrousdichloromethane (30 mL) at −20° C. The solution was allowed to stir atroom temperature for 2 h. To the obtained mixture,2-[(2-hydroxyethyl)oxy]ethyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside,2, (3.36 g, 7.7 mmol) and 1H-tetrazole (0.45 M in MeCN, 10.0 mL, 6.0mmol) were added followed by stirring at room temperature for 14 h. Thereaction mixture was quenched by addition of excess 5% aqueous NaHCO₃.The emulsion was diluted with 5% aqueous NaHCO₃ (50 mL), and the productwas extracted with ethyl acetate (3×100 mL). Extracts were washed withbrine, dried over anhydrous Na₂SO₄, and evaporated to oil. The productwas isolated chromatographically on a silica gel column eluted with astep gradient from ethyl acetate—TEA—hexane (20:5:75) to TEA—ethylacetate (5:95). Collected fractions were evaporated to give the titlecompound as an off-white foam (5.75 g, 71.2%). ³¹P NMR (mixture ofdiastereomers, CD₃CN): δ 148.91, 148.38.

Example 22N²-Phenoxyacetyl-5′-O-(4,4′-dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[2-[[2-[(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)oxy]ethyl]oxy]ethoxy]phosphinyl]-2′-deoxyguanosine (19b)

Solution of bis(N,N-diisopropylamino) chlorophosphite (1.80 g, 6.75mmol) in anhydrous dichloromethane (10 mL) was added dropwise to astirred solution ofN²-phenoxyacetyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyguanosine, 11b,(3.80 g, 5.4 mmol, Rasayan, Inc., Encinitas, Calif.) andN-ethyl-N,N-diisopropylamine (1.33 g, 10.3 mmol) in anhydrousdichloromethane (20 mL) at −20° C. The solution was allowed to stir atroom temperature for 2 h. To the obtained mixture,2-[(2-hydroxyethyl)oxy]ethyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside,2, (2.71 g, 6.2 mmol) and 1H-tetrazole (0.45 M in MeCN, 4.8 mL) wereadded followed by stirring at room temperature for 14 h. The reactionmixture was quenched by addition of excess 5% aqueous NaHCO₃. Theemulsion was diluted with 5% aqueous NaHCO₃ (50 mL), and the product wasextracted with ethyl acetate (3×100 mL). Extracts were washed withbrine, dried over anhydrous Na₂SO₄, and evaporated to oil. The productwas isolated chromatographically on a silica gel column eluted with astep gradient from ethyl acetate—TEA—hexane (20:5:75) to TEA—ethylacetate (5:95). Collected fractions were evaporated to give the titlecompound as an off-white foam (4.58 g, 66.8%). ³¹P NMR (mixture ofdiastereomers, CD₃CN): δ 149.04, 148.60.

Example 23N²-(4-i-Propylphenoxy)acetyl-5′-O-(4,4′-dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[2-[[2-[(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)oxy]ethyl] oxy]ethoxy]phosphinyl]-2′-deoxyguanosine (19c)

Solution of bis(N,N-diisopropylamino) chlorophosphite (1.97 g, 7.38mmol) in anhydrous dichloromethane (10 mL) was added dropwise to astirred solution of N²-(4-i-propylphenoxy)acetyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyguanosine, 11c, (4.40 g, 5.9mmol, Rasayan, Inc., Encinitas, Calif.) and N-ethyl-N,N-diisopropylamine(1.45 g, 11.2 mmol) in anhydrous dichloromethane (20 mL) at −20° C. Thesolution was allowed to stir at room temperature for 2 h. To theobtained mixture, 2-[(2-hydroxyethyl)oxy]ethyl2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside, 2, (2.96 g, 6.8 mmol) and1H-tetrazole (0.45 M in MeCN, 5.3 mL) were added followed by stirring atroom temperature for 14 h. The reaction mixture was quenched by additionof excess 5% aqueous NaHCO₃. The emulsion was diluted with 5% aqueousNaHCO₃ (50 mL), and the product was extracted with ethyl acetate (3×100mL). Extracts were washed with brine, dried over anhydrous Na₂SO₄, andevaporated to oil. The product was isolated chromatographically on asilica gel column eluted with a step gradient from ethylacetate—TEA—hexane (20:5:75) to TEA—ethyl acetate (5:95). Collectedfractions were evaporated to give the title compound as an off-whitefoam (4.84 g, 62.6%). ³¹P NMR (mixture of diastereomers, CD₃CN): δ148.75, 148.19.

Example 24N²—(N,N-dimethylformamidino)-5′-O-(4,4′-dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[2-[[2-[(2,3,4,6-tetra-O-acetyl-13-D-glucopyranosyl)oxy]ethyl]oxy]ethoxy]phosphinyl]-2′-deoxyguanosine (19d)

Solution of bis(N,N-diisopropylamino) chlorophosphite (1.73 g, 6.50mmol) in anhydrous dichloromethane (10 mL) was added dropwise to astirred solution ofN²—(N,N-dimethylformamidino)-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyguanosine,11d, (3.25 g, 5.20 mmol, Rasayan, Inc., Encinitas, Calif.) andN-ethyl-N,N-diisopropylamine (1.28 g, 9.88 mmol) in anhydrousdichloromethane (20 mL) at −20° C. The solution was allowed to stir atroom temperature for 2 h. To the obtained mixture,2-[(2-hydroxyethyl)oxy]ethyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside,2, (2.61 g, 5.98 mmol) and 1H-tetrazole (0.45 M in MeCN, 4.6 mL) wereadded followed by stirring at room temperature for 14 h. The reactionmixture was quenched by addition of excess 5% aqueous NaHCO₃. Theemulsion was diluted with 5% aqueous NaHCO₃ (50 mL), and the product wasextracted with ethyl acetate (3×100 mL). Extracts were washed withbrine, dried over anhydrous Na₂SO₄, and evaporated to oil. The productwas isolated chromatographically on a silica gel column eluted with astep gradient from ethyl acetate—TEA—hexane (20:5:75) to TEA—ethylacetate (5:95). Collected fractions were evaporated to give the titlecompound as an off-white foam (4.34 g, 70.1%). ³¹P NMR (mixture ofdiastereomers, CD₃CN): δ 148.63, 148.08.

Example 255′-O-(4,4′-Dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[2-[[2-[(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)oxy]ethyl]oxy]ethoxy]phosphinyl]thymidine(20)

Solution of bis(N,N-diisopropylamino) chlorophosphite (1.28 g, 4.79mmol) in anhydrous dichloromethane (5 mL) was added dropwise to astirred solution of 5′-O-(4,4′-dimethoxytrityl)thymidine, 8, (2.08 g,3.83 mmol) and N-ethyl-N,N-diisopropylamine (0.94 g, 7.28 mmol) inanhydrous dichloromethane (15 mL) at −20° C. The solution was allowed tostir at room temperature for 2 h. To the obtained mixture,2-[(2-hydroxyethyl)oxy]ethyl2,3,4,6-tetra-O-acetyl-β-D-galactopyranoside, 3, (1.92 g, 4.40 mmol,prepared as disclosed in Karjala, S. and Link, K. P., J. Amer. Chem.Soc., 1940, 62, 917-922) and 1H-tetrazole (0.45 M in MeCN, 3.4 mL) wereadded followed by stirring at room temperature for 14 h. The reactionmixture was quenched by addition of excess 5% aqueous NaHCO₃. Theemulsion was diluted with 5% aqueous NaHCO₃ (50 mL), and the product wasextracted with ethyl acetate (3×100 mL). Extracts were washed withbrine, dried over anhydrous Na₂SO₄, and evaporated to oil. The productwas isolated chromatographically on a silica gel column as describedabove for the respective β-D-glucopyranoside analog 16 to give the titlecompound (3.60 g, 84.8%). ³¹P NMR (mixture of diastereomers, CD₃CN): δ148.66, 148.09.

Example 26N⁴-Benzoyl-5′-O-(4,4′-dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[2-[[2-[(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)oxy]ethyl]oxy]ethoxy]phosphinyl]-2′-deoxycytidine (21a)

Solution of bis(N,N-diisopropylamino) chlorophosphite (1.28 g, 4.79mmol) in anhydrous dichloromethane (5 mL) was added dropwise to astirred solution ofN⁴-benzoyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxycytidine, 9a, (2.08 g,3.73 mmol) and N-ethyl-N,N-diisopropylamine (1.01 g, 7.83 mmol) inanhydrous dichloromethane (15 mL) at −20° C. The solution was allowed tostir at room temperature for 2 h. To the obtained mixture,2-[(2-hydroxyethyl)oxy]ethyl2,3,4,6-tetra-O-acetyl-β-D-galactopyranoside, 3, (2.07 g, 4.74 mmol) and1H-tetrazole (0.45 M in MeCN, 3.7 mL) were added followed by stirring atroom temperature for 14 h. The reaction mixture was quenched by additionof excess 5% aqueous NaHCO₃. The emulsion was diluted with 5% aqueousNaHCO₃ (50 mL), and the product was extracted with ethyl acetate (3×100mL). Extracts were washed with brine, dried over anhydrous Na₂SO₄, andevaporated to oil. The product was isolated chromatographically on asilica gel column as described above for the respectiveβ-D-glucopyranoside analog 17a give the title compound (3.97 g, 80.3%).³¹P NMR (mixture of diastereomers, CD₃CN): δ 148.69, 148.33.

Example 27N⁴-Acetyl-5′-O-(4,4′-dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[2-[[2-[(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)oxy]ethyl]oxy] ethoxy]phosphinyl]-2′-deoxycytidine (21c)

Solution of bis(N,N-diisopropylamino) chlorophosphite (1.48 g, 5.35mmol) in anhydrous dichloromethane (5 mL) was added dropwise to astirred solution ofN⁴-acetyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxycytidine, 9c, (2.45 g,4.28 mmol) and N-ethyl-N,N-diisopropylamine (1.05 g, 8.13 mmol) inanhydrous dichloromethane (15 mL) at −20° C. The solution was allowed tostir at room temperature for 2 h. To the obtained mixture,2-[(2-hydroxyethyl)oxy]ethyl2,3,4,6-tetra-O-acetyl-β-D-galactopyranoside, 3, (2.15 g, 4.92 mmol) and1H-tetrazole (0.45 M in MeCN, 3.8 mL) were added followed by stirring atroom temperature for 14 h. The reaction mixture was quenched by additionof excess 5% aqueous NaHCO₃. The emulsion was diluted with 5% aqueousNaHCO₃ (50 mL), and the product was extracted with ethyl acetate (3×100mL). Extracts were washed with brine, dried over anhydrous Na₂SO₄, andevaporated to oil. The product was isolated chromatographically on asilica gel column as described above for the respectiveβ-D-glucopyranoside analog 17c to give the title compound (4.05 g,83.1%). ³¹P NMR (mixture of diastereomers, CD₃CN): δ 149.16, 148.66.

Example 28N⁶-Benzoyl-5′-O-(4,4′-dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[2-[[2-[(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)oxy]ethyl] oxy] ethoxy]phosphinyl]-2′-deoxyadenosine (22a)

Solution of bis(N,N-diisopropylamino) chlorophosphite (1.32 g, 4.94mmol) in anhydrous dichloromethane (5 mL) was added dropwise to astirred solution ofN⁶-benzoyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyadenosine, 10a, (2.60 g,3.95 mmol) and N-ethyl-N,N-diisopropylamine (0.97 g, 7.51 mmol) inanhydrous dichloromethane (15 mL) at −20° C. The solution was allowed tostir at room temperature for 2 h. To the obtained mixture,2-[(2-hydroxyethyl)oxy]ethyl2,3,4,6-tetra-O-acetyl-β-D-galactopyranoside, 3, (1.98 g, 4.54 mmol) and1H-tetrazole (0.45 M in MeCN, 3.5 mL) were added followed by stirring atroom temperature for 14 h. The reaction mixture was quenched by additionof excess 5% aqueous NaHCO₃. The emulsion was diluted with 5% aqueousNaHCO₃ (50 mL), and the product was extracted with ethyl acetate (3×100mL). Extracts were washed with brine, dried over anhydrous Na₂SO₄, andevaporated to oil. The product was isolated chromatographically on asilica gel column as described above for the respectiveβ-D-glucopyranoside analog 18a give the title compound (3.65 g, 75.5%).³¹P NMR (mixture of diastereomers, CD₃CN): δ 148.73, 148.18.

Example 29N⁶-Phenoxyacetyl-5′-O-(4,4′-dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[2-[[2-[(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)oxy]ethyl]oxy] ethoxy]phosphinyl]-2′-deoxyadenosine (22b)

Solution of bis(N,N-diisopropylamino) chlorophosphite (1.25 g, 4.68mmol) in anhydrous dichloromethane (5 mL) was added dropwise to astirred solution ofN⁶-phenoxyacetyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyadenosine, 10b,(2.57 g, 3.74 mmol) and N-ethyl-N,N-diisopropylamine (0.92 g, 7.1 mmol)in anhydrous dichloromethane (15 mL) at −20° C. The solution was allowedto stir at room temperature for 2 h. To the obtained mixture,2-[(2-hydroxyethyl)oxy]ethyl2,3,4,6-tetra-O-acetyl-β-D-galactopyranoside, 3, (1.88 g, 4.30 mmol) and1H-tetrazole (0.45 M in MeCN, 3.4 mL) were added followed by stirring atroom temperature for 14 h. The reaction mixture was quenched by additionof excess 5% aqueous NaHCO₃. The emulsion was diluted with 5% aqueousNaHCO₃ (50 mL), and the product was extracted with ethyl acetate (3×100mL). Extracts were washed with brine, dried over anhydrous Na₂SO₄, andevaporated to oil. The product was isolated chromatographically on asilica gel column as described above for the respectiveβ-D-glucopyranoside analog 18b to give the title compound (3.30 g,70.4%). ³¹P NMR (mixture of diastereomers, CD₃CN): δ 148.80, 148.17.

Example 30N²-Isobutyryl-5′-O-(4,4′-dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[2-[[2-[(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)oxy]ethyl]oxy]ethoxy]phosphinyl]-2′-deoxyguanosine (23a)

Solution of bis(N,N-diisopropylamino) chlorophosphite (1.33 g, 5.0 mmol)in anhydrous dichloromethane (15 mL) was added dropwise to a stirredsolution of N²-isobutyryl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyguanosine,11a, (2.56 g, 4.0 mmol, Rasayan, Inc., Encinitas, Calif.) andN-ethyl-N,N-diisopropylamine (098 g, 7.60 mmol) in anhydrousdichloromethane (30 mL) at −20° C. The solution was allowed to stir atroom temperature for 2 h. To the obtained mixture,2-[(2-hydroxyethyl)oxy]ethyl2,3,4,6-tetra-O-acetyl-β-D-galactopyranoside, 3, (2.01 g, 4.60 mmol) and1H-tetrazole (0.45 M in MeCN, 3.6 mL) were added followed by stirring atroom temperature for 14 h. The reaction mixture was quenched by additionof excess 5% aqueous NaHCO₃. The emulsion was diluted with 5% aqueousNaHCO₃ (50 mL), and the product was extracted with ethyl acetate (3×100mL). Extracts were washed with brine, dried over anhydrous Na₂SO₄, andevaporated to oil. The product was isolated chromatographically on asilica gel column as described above for the respectiveβ-D-glucopyranoside analog 19a to give the title compound as anoff-white foam (3.56 g, 73.9%). ³¹P NMR (mixture of diastereomers,CD₃CN): δ 149.32, 148.66.

Example 31N²-Phenoxyacetyl-5′-O-(4,4′-dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[2-[[2-[(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)oxy]ethyl] oxy]ethoxy]phosphinyl]-2′-deoxyguanosine (23b)

Solution of bis(N,N-diisopropylamino) chlorophosphite (1.22 g, 4.59mmol) in anhydrous dichloromethane (10 mL) was added dropwise to astirred solution ofN²-phenoxyacetyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyguanosine, 11b,(2.58 g, 3.67 mmol, Rasayan, Inc., Encinitas, Calif.) andN-ethyl-N,N-diisopropylamine (0.90 g, 6.97 mmol) in anhydrousdichloromethane (20 mL) at −20° C. The solution was allowed to stir atroom temperature for 2 h. To the obtained mixture,2-[(2-hydroxyethyl)oxy]ethyl2,3,4,6-tetra-O-acetyl-β-D-galactopyranoside, 3, (1.84 g, 4.22 mmol) and1H-tetrazole (0.45 M in MeCN, 3.3 mL) were added followed by stirring atroom temperature for 14 h. The reaction mixture was quenched by additionof excess 5% aqueous NaHCO₃. The emulsion was diluted with 5% aqueousNaHCO₃ (50 mL), and the product was extracted with ethyl acetate (3×100mL). Extracts were washed with brine, dried over anhydrous Na₂SO₄, andevaporated to oil. The product was isolated chromatographically on asilica gel column as described above for the respectiveβ-D-glucopyranoside analog 19b to give the title compound as anoff-white foam (3.23 g, 69.3%). ³¹P NMR (mixture of diastereomers,CD₃CN): δ 149.33, 148.69.

Example 321-O-(3-Hydroxypropyl)-3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-glucopyranoside(6)

A mixture of compound 4 (8.80 g, 26.7 mmol, prepared as disclosed inFukase, K.; Ueno, A.; Fukase, Y; et al., Bull. Chem. Soc. Japan 2003,76, 485-500), 1,3-propanediol (30.5 g, 0.4 mol), and pyridiniump-toluenesulfonate (5.37 g, 21.4 mmol) was heated at 80° C. for 18 h,then distributed between DCM (50 mL) and a 1:1 mixture of brine and 5%aqueous NaHCO₃ (100 mL). The organic phase was separated, dried overNa₂SO₄, and evaporated in vacuo. The product was isolated by columnchromatography on silica gel eluted with a step gradient from DCM to amixture of MeOH and DCM (8:92). The solid obtained by evaporation of therelevant fractions was re-crystallized from a mixture of methyl t-butylether and DCM to give 2.91 g of pure ?? as an off-white solid. Found: C,49.69; H, 6.77; N, 3.57. C₁₇H₂₇N₁₀.½H₂O. Calculated: C, 49.27; H, 6.81;N, 3.38. ¹H NMR (CD₃CN): δ 6.395 (1H, br. d, J=9.5 Hz); 5.134 (1H, dd,J=10.5, 9.5 Hz); 4.932 (1H, t, J=9.7 Hz); 4.612 (1H, d, J=8.5 Hz); 4.215(1H, dd, J=12.5, 5.0 Hz); 4.074 (1H, dd, J=12.5, 2.5 Hz); 3.876-3.835(1H, m); 3.802-3.759 (2H, m); 3.625-3.560 (1H, m); 3.560-3.523 (2H, m);2.554 (1H, t, J=5.2 Hz); 2.015 (3H, s); 1.965 (3H, s); 1.935 (3H, s);1.820 (3H, s); 1.707 (2H, pent., J=6.2 Hz). The mother liquor wasevaporated, and the residue was re-crystallized from the same mixture togive additional product (2.61 g) in a total yield of 51.3%.

Example 335′-O-(4,4′-Dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[3-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-glucopyranosyl)oxy]propoxy]phosphinyl]thymidine(24)

To the magnetically stirred mixture of5′-O-(4,4′-dimethoxytrityl)thymidine, 8, (5.99 g, 11.0 mmol),N-ethyl-N,N-diisopropylamine (2.70 g, 20.90 mmol), and anhydrous CH₃CN(30 mL) was added a solution of N,N,N′,N′-tetraisopropyl chlorophosphite(3.67 g, 13.75 mmol) in anhydrous DCM (10 mL) at −20° C. The mixture wasallowed to warm up to room temperature over 2 h. A solution of1-O-(3-hydroxypropyl)-3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-glucopyranoside,6, (0.405 g, 1 mmol) in anhydrous acetonitrile (5 mL) was added followedby the addition of a solution of 1H-tetrazole in acetonitrile (0.45 M,9.8 mL). The reaction mixture was stirred for 18 h at room temperatureand was quenched by adding 5% aqueous NaHCO₃. The product was extractedwith ethyl acetate (3×100 mL). Combined extracts were washed with brine(50 mL), dried over anhydrous Na₂SO₄, and evaporated to dryness. Theproduct was isolated by column chromatography on silica gel eluted witha step gradient starting from TEA—ethyl acetate—hexane (5:20:75) to amixture of TEA in ethyl acetate (10:90). Evaporation of fractions gavecompound 24 as white solid foam in a yield of 10.37 g (87.4%). ³¹P NMR(CD₃CN): δ 147.60, 147.57. ¹H NMR (CD₃CN): δ 8.955 (1H, br. s);7.45-7.41 (3H, m); 7.35-7.28 (6H, m); 7.28-7.21 (1H, m); 6.90-6.84 (4H,m); 6.590 (0.5H, d, J=9.0 Hz); 6.342 (0.5H, d, J=4.5 Hz); 6.230 (0.5H,t, J=9.0 Hz); 6.195 (0.5H, t, J=7.0 Hz); 5.184 (0.5H, dd, J=10.5, 9.5Hz); 5.135 (0.5H, dd, J=10.5, 9.5 Hz); 4.920 (1H, q, J=9.7 Hz);4.630-4.525 (2H, m); 4.188 (1H, ddd, J=12.5, 12.5, 5.0 Hz); 4.13-4.09(0.5H, m); 4.09-3.95 (2H, m); 3.89-3.805 (0.5H, m); 3.771 (3H, s); 3.765(3H, s); 3.805-3.62 (3H, m); 3.62-3.475 (4H, m); 3.39-3.23 (2H, m);2.46-230 (2H, m); 1.982, 1.965, 1.962, 1.954, 1.940, 1.935 (total 9H,singlets); 1.789 (1.5H, s); 1.777 (1.5H, s); 1.691 (2H, pent., J=6.5Hz); 1.553 (1.5H, d, J=1.0 Hz); 1.490 (1.5H, d, J=1.0 Hz); 1.18-1.11(9H, m); 1.040 (3H, d, J=7.0 Hz).

Example 34N⁴-Benzoyl-5′-O-(4,4′-dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[3-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-glucopyranosyl)oxy]propoxy]phosphinyl]-2′-deoxycytidine(25a)

Solution of bis(N,N-diisopropylamino) chlorophosphite (3.20 g, 12.0mmol) in anhydrous dichloromethane (15 mL) was added dropwise to astirred solution ofN⁴-benzoyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxycytidine, 9a, (6.08 g,9.6 mmol) and N-ethyl-N,N-diisopropylamine (2.36 g, 18.2 mmol) inanhydrous dichloromethane (30 mL) at −20° C. The solution was allowed tostir at room temperature for 2 h. To the obtained mixture,1-O-(3-hydroxypropyl)-3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-glucopyranoside,6, (4.48 g, 11.0 mmol) and 1H-tetrazole (0.45 M in MeCN, 8.5 mL) wereadded followed by stirring at room temperature for 14 h. The reactionmixture was quenched by addition of excess 5% aqueous NaHCO₃. Theemulsion was diluted with 5% aqueous NaHCO₃ (50 mL), and the product wasextracted with ethyl acetate (3×100 mL). Extracts were washed withbrine, dried over anhydrous Na₂SO₄, and evaporated to oil. The productwas isolated by column chromatography on silica gel eluted with a stepgradient starting from TEA—ethyl acetate—hexane (5:20:75) to a mixtureof TEA in ethyl acetate (10:90). Evaporation of fractions gave thetarget compound as white solid foam in a yield of 9.10 g (81.1%). ³¹PNMR (mixture of diastereomers, CD₃CN): δ 147.63, 147.21.

Example 35N⁴-Acetyl-5′-O-(4,4′-dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[3-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-glucopyranosyl)oxy]propoxy]phosphinyl]-2′-deoxycytidine(25c)

Solution of bis(N,N-diisopropylamino) chlorophosphite (1.67 g, 6.25mmol) in anhydrous dichloromethane (5 mL) was added dropwise to astirred solution ofN⁴-acetyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxycytidine, 9c, (2.86 g, 5.0mmol) and N-ethyl-N,N-diisopropylamine (12.3 g, 9.5 mmol) in anhydrousdichloromethane (15 mL) at −20° C. The solution was allowed to stir atroom temperature for 2 h. To the obtained mixture,1-O-(3-hydroxypropyl)-3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-glucopyranoside,6, (2.33 g, 5.75 mmol) and 1H-tetrazole (0.45 M in MeCN, 4.5 mL) wereadded followed by stirring at room temperature for 14 h. The reactionmixture was quenched by addition of excess 5% aqueous NaHCO₃. Theemulsion was diluted with 5% aqueous NaHCO₃ (50 mL), and the product wasextracted with ethyl acetate (3×100 mL). Extracts were washed withbrine, dried over anhydrous Na₂SO₄, and evaporated to oil. The productwas isolated by column chromatography on silica gel eluted with a stepgradient starting from TEA—ethyl acetate—hexane (5:20:75) to a mixtureof TEA in ethyl acetate (10:90). Evaporation of fractions gave the titlecompound as white solid foam in a yield of 4.14 g (74.9%). ³¹P NMR(mixture of diastereomers, CD₃CN): δ 149.43, 148.77.

Example 36N⁶-Benzoyl-5′-O-(4,4′-dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[3-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-glucopyranosyl)oxy]propoxy]phosphinyl]-2′-deoxyadenosine(26a)

Solution of bis(N,N-diisopropylamino) chlorophosphite (1.77 g, 6.63mmol) in anhydrous dichloromethane (5 mL) was added dropwise to astirred solution ofN⁶-benzoyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyadenosine, 10a, (3.49 g,5.3 mmol) and N-ethyl-N,N-diisopropylamine (1.30 g, 10.1 mmol) inanhydrous dichloromethane (15 mL) at −20° C. The solution was allowed tostir at room temperature for 2 h. To the obtained mixture,1-O-(3-hydroxypropyl)-3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-glucopyranoside,6, (2.47 g, 6.10 mmol) and 1H-tetrazole (0.45 M in MeCN, 4.7 mL) wereadded followed by stirring at room temperature for 14 h. The reactionmixture was quenched by addition of excess 5% aqueous NaHCO₃. Theemulsion was diluted with 5% aqueous NaHCO₃ (50 mL), and the product wasextracted with ethyl acetate (3×100 mL). Extracts were washed withbrine, dried over anhydrous Na₂SO₄, and evaporated to oil. Evaporationof fractions gave the title compound as white solid foam in a yield of4.47 g (70.7%). ³¹P NMR (mixture of diastereomers, CD₃CN): δ 148.58,148.11.

Example 37N⁶-Phenoxyacetyl-5′-O-(4,4′-dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[3-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-glucopyranosyl)oxy]propoxy]phosphinyl]-2′-deoxyadenosine(26b)

Solution of bis(N,N-diisopropylamino) chlorophosphite (1.57 g, 5.88mmol) in anhydrous dichloromethane (5 mL) was added dropwise to astirred solution ofN⁶-phenoxyacetyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyadenosine, 10b,(3.23 g, 4.70 mmol) and N-ethyl-N,N-diisopropylamine (1.15 g, 8.9 mmol)in anhydrous dichloromethane (15 mL) at −20° C. The solution was allowedto stir at room temperature for 2 h. To the obtained mixture,1-O-(3-hydroxypropyl)-3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-glucopyranoside,6, (2.19 g, 5.41 mmol) and 1H-tetrazole (0.45 M in MeCN, 4.2 mL) wereadded followed by stirring at room temperature for 14 h. The reactionmixture was quenched by addition of excess 5% aqueous NaHCO₃. Theemulsion was diluted with 5% aqueous NaHCO₃ (50 mL), and the product wasextracted with ethyl acetate (3×100 mL). Extracts were washed withbrine, dried over anhydrous Na₂SO₄, and evaporated to oil. Evaporationof fractions gave the title compound as white solid foam in a yield of3.92 g (68.2%). ³¹P NMR (mixture of diastereomers, CD₃CN): δ 148.98,148.29.

Example 38N²-Isobutyryl-5′-O-(4,4′-dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[3-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-glucopyranosyl)oxy]propoxy]phosphinyl]-2′-deoxyguanosine(27a)

Solution of bis(N,N-diisopropylamino) chlorophosphite (2.23 g, 8.38mmol) in anhydrous dichloromethane (15 mL) was added dropwise to astirred solution ofN²-isobutyryl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyguanosine, 11a, (4.29g, 6.70 mmol) and N-ethyl-N,N-diisopropylamine (1.64 g, 12.73 mmol) inanhydrous dichloromethane (30 mL) at −20° C. The solution was allowed tostir at room temperature for 2 h. To the obtained mixture,1-O-(3-hydroxypropyl)-3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-glucopyranoside,6, (3.12 g, 7.71 mmol) and 1H-tetrazole (0.45 M in MeCN, 6.0 mL) wereadded followed by stirring at room temperature for 14 h. The reactionmixture was quenched by addition of excess 5% aqueous NaHCO₃. Theemulsion was diluted with 5% aqueous NaHCO₃ (50 mL), and the product wasextracted with ethyl acetate (3×100 mL). Extracts were washed withbrine, dried over anhydrous Na₂SO₄, and evaporated to oil. Evaporationof fractions gave the title compound as white solid foam in a yield of5.68 g (72.2%). ³¹P NMR (mixture of diastereomers, CD₃CN): δ 148.91,148.33.

Example 39N²-Phenoxyacetyl-5′-O-(4,4′-dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[3-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-glucopyranosyl)oxy]propoxy]phosphinyl]-2′-deoxyguanosine(27b)

Solution of bis(N,N-diisopropylamino) chlorophosphite (1.50 g, 5.63mmol) in anhydrous dichloromethane (10 mL) was added dropwise to astirred solution ofN²-phenoxyacetyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyguanosine, 11b,(3.17 g, 4.50 mmol) and N-ethyl-N,N-diisopropylamine (1.10 g, 8.55 mmol)in anhydrous dichloromethane (20 mL) at −20° C. The solution was allowedto stir at room temperature for 2 h. To the obtained mixture,1-O-(3-hydroxypropyl)-3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-glucopyranoside,6, (2.10 g, 5.18 mmol) and 1H-tetrazole (0.45 M in MeCN, 4.0 mL) wereadded followed by stirring at room temperature for 14 h. The reactionmixture was quenched by addition of excess 5% aqueous NaHCO₃. Theemulsion was diluted with 5% aqueous NaHCO₃ (100 mL), and the productwas extracted with ethyl acetate (3×70 mL). Extracts were washed withbrine, dried over anhydrous Na₂SO₄, and evaporated to oil. Evaporationof fractions gave the title compound as white solid foam in a yield of3.66 g (65.7%). ³¹P NMR (mixture of diastereomers, CD₃CN): δ 148.72,148.27.

Example 401-O-(5-Hydroxy-3-oxapentyl)-3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranoside(7b)

A mixture of compound 5 (13.17 g, 40.0 mmol, prepared as disclosed inFukase, K.; Ueno, A.; Fukase, Y; et al., Bull. Chem. Soc. Japan 2003,76, 485-500), anhydrous diethyleneglycol (42.5 g, 0.4 mol), andpyridinium p-toluenesulfonate (9.05 g, 36.0 mmol) was heated at 80° C.for 18 h, then distributed between DCM (300 mL) and a 1:1 mixture ofbrine and 5% aqueous NaHCO₃ (100 mL). The organic phase was separated,dried over Na₂SO₄, and evaporated in vacuo. The product was isolated bycolumn chromatography on silica gel eluted with a step gradient from DCMto a mixture of MeOH and DCM (8:92). Evaporation of the relevantfractions gave the title compound as an off-white solid (7.92 g, 45.4%).¹H NMR (CD₃CN): δ 6.572 (1H, d, J=9.5 Hz); 5.278 (1H, d, J=3 Hz); 5.010(1H, dd, J=11.5 Hz, J=3 Hz); 4.673 (1H, d, J=8.5 Hz); 4.109 (1H, dd, J=9Hz, J=7 Hz); 4.066 (1H, dd, J=9 Hz, J=6 Hz); 3.951 (2H, m); 3.828 (1H,m); 3.703 (1H, m); 3.64-3.54 (4H, m); 3.54-3.45 (2H, m); 3.053 (1H, br.s); 2.098 (3H, s) 1.988 (3H, s); 1.910 (3H, s), 1.845 (3H, s). ES MS:436.1 (MH⁺). Calculated for C₁₈H₂₉NO₁₁H⁺: 436.2.

Example 415′-O-(4,4′-Dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[5-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3-oxapentyloxy]phosphinyl]thymidine(28)

To the magnetically stirred mixture of5′-O-(4,4′-dimethoxytrityl)thymidine, 8, (1.142 g, 2.10 mmol),N-ethyl-N,N-diisopropylamine (0.515 g, 3.99 mmol), and anhydrous CH₃CN(10 mL) was added a solution of N,N′,N′-tetraisopropyl chlorophosphite(0.700 g, 2.63 mmol) in anhydrous DCM (5 mL) at −20° C. The mixture wasallowed to warm up to room temperature over 2 h. A solution of1-O-(5-hydroxy-3-oxapentyl)-3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranoside,7b, (1.097 g, 2.52 mmol) in anhydrous acetonitrile (5 mL) was addedfollowed by the addition of a solution of 1H-tetrazole in acetonitrile(0.45 M, 1.87 mL). The reaction mixture was stirred for 18 h at roomtemperature and was quenched by adding 5% aqueous NaHCO₃. The productwas extracted with ethyl acetate (3×50 mL). Combined extracts werewashed with brine (50 mL), dried over anhydrous Na₂SO₄, and evaporatedto dryness. The product was isolated by column chromatography on silicagel eluted with a step gradient starting from TEA—ethyl acetate—hexane(5:20:75) to a mixture of TEA in ethyl acetate (10:90). Evaporation offractions gave compound 24 as white solid foam in a yield of 1.760 g(75.6%).

Fast diastereomer: ¹H NMR (CD₃CN): δ 9.09 (1H, br. s); 7.5-7.4 (3H, m);7.375-7.30 (6H, m); 7.30-7.23 (1H, m); 6.9-6.8 (4H, m); 6.335 (1H, d,J=9 Hz); 6.248 (1H, t, J=7 Hz); 5.31-5.27 (1H, m); 5.025 (1H, dd, J=11.2Hz, J=3.5 Hz); 4.66-4.59 (1H, m); 4.565 (1H, d, J=8.5 Hz); 4.15-4.11(1H, m); 4.11-4.03 (2H, m); 3.99-3.89 (2H, m); 3.84-3.70 (2H, m); 3.783and 3.766 (total 6H, s, s); 3.67-3.53 (5H, m); 3.53-3.43 (4H, m); 3.355(1H, dd, J=10.5 Hz, J=3.0 Hz); (1H, dd, J=10.5 Hz, J=3.5 Hz); 2.41-2.33(2H, m); 2.090 (3H, s); 1.968 (3H, s), 1.913 (3H, s); 1.820 (3H, s);1.480 (3H, d, J=0.5 Hz); 1.149 (12H, t, J=6.5 Hz). ³¹P NMR (CD₃CN): δ149.99.

Slow diastereomer: ¹H NMR (CD₃CN): δ 8.976 (1H, br. s); 7.5-7.4 (3H, m);7.375-7.30 (6H, m); 7.30-7.23 (1H, m); 6.9-6.8 (4H, m); 6.358 (1H, d,J=9.5 Hz); 6.242 (1H, t, J=7 Hz); 5.29-5.27 (1H, m); 5.030 (1H, dd,J=11.5 Hz, J=3.5 Hz); 4.64-4.54 (2H, m); 4.15-4.03 (3H, m); 3.99-3.89(2H, m); 3.85-3.79 (1H, m); 3.814 and 3.764 (total 6H, s, s); 3.78-3.70(1H, m); 3.70-3.61 (2H, m); 3.61-3.48 (5H, m); 3.35-3.255 (1H, m);2.46-2.39 (1H, m); 2.37-2.29 (1H, m); 2.094 (3H, s); 1.974 (3H, s),1.913 (3H, s); 1.832 (3H, s); 1.498 (3H, s); 1.143 (6H, d, J=6.5 Hz);1.051 (6H, d, J=6.5 Hz). ³¹P NMR (CD₃CN): δ 149.67.

Example 42N⁴-Benzoyl-5′-O-(4,4′-dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[5-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3-oxapentyloxy]phosphinyl]-2′-deoxycytidine(29a)

Solution of bis(N,N-diisopropylamino) chlorophosphite (667 mg, 2.50mmol) in anhydrous dichloromethane (5 mL) was added dropwise to astirred solution ofN⁴-benzoyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxycytidine, 9a, (1267 mg,2.0 mmol) and N-ethyl-N,N-diisopropylamine (491 mg, 3.80 mmol) inanhydrous dichloromethane (10 mL) at −20° C. The solution was allowed tostir at room temperature for 2 h. To the obtained mixture, compound 7b(1045 mg, 2.40 mmol) and 1H-tetrazole (0.45 M in MeCN, 1.8 mL) wereadded followed by stirring at room temperature for 14 h. The reactionmixture was quenched by addition of excess 5% aqueous NaHCO₃. Theemulsion was diluted with 5% aqueous NaHCO₃ (30 mL), and the product wasextracted with ethyl acetate (3×50 mL). Extracts were washed with brine,dried over anhydrous Na₂SO₄, and evaporated to oil. The product wasisolated by column chromatography on silica gel eluted with a stepgradient starting from TEA—ethyl acetate—hexane (5:20:75) to a mixtureof TEA in ethyl acetate (10:90). Evaporation of fractions gave thetarget compound as white solid foam in a yield of 1769 mg (73.8%). ³¹PNMR (mixture of diastereomers, CD₃CN): δ 148.91, 148.37.

Example 43N⁴-Acetyl-5′-O-(4,4′-dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[5-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3-oxapentyloxy]phosphinyl]-2′-deoxycytidine(29c)

Solution of bis(N,N-diisopropylamino) chlorophosphite (767 mg, 2.88mmol) in anhydrous dichloromethane (5 mL) was added dropwise to astirred solution ofN⁴-acetyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxycytidine, 9c, (1315 mg,2.30 mmol) and N-ethyl-N,N-diisopropylamine (564 mg, 4.37 mmol) inanhydrous dichloromethane (10 mL) at −20° C. The solution was allowed tostir at room temperature for 2 h. To the obtained mixture, compound 7b(1202 mg, 2.76 mmol) and 1H-tetrazole (0.45 M in MeCN, 2.1 mL) wereadded followed by stirring at room temperature for 14 h. The reactionmixture was quenched by addition of excess 5% aqueous NaHCO₃. Theemulsion was diluted with 5% aqueous NaHCO₃ (30 mL), and the product wasextracted with ethyl acetate (3×50 mL). Extracts were washed with brine,dried over anhydrous Na₂SO₄, and evaporated to oil. The product wasisolated by column chromatography on silica gel eluted with a stepgradient starting from TEA—ethyl acetate—hexane (5:20:75) to a mixtureof TEA in ethyl acetate (10:90). Evaporation of fractions gave the titlecompound as white solid foam in a yield of 1835 mg (70.2%). ³¹P NMR(mixture of diastereomers, CD₃CN): δ 148.78, 148.11.

Example 44N⁶-Benzoyl-5′-O-(4,4′-dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[5-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3-oxapentyloxy]phosphinyl]-2′-deoxyadenosine(30a)

Solution of bis(N,N-diisopropylamino) chlorophosphite (750 mg, 2.81mmol) in anhydrous dichloromethane (5 mL) was added dropwise to astirred solution ofN⁶-benzoyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyadenosine, 10a, (1480,2.25 mmol) and N-ethyl-N,N-diisopropylamine (552 mg, 4.28 mmol) inanhydrous dichloromethane (10 mL) at −20° C. The solution was allowed tostir at room temperature for 2 h. To the obtained mixture, compound 7b(1176 mg, 2.70 mmol) and 1H-tetrazole (0.45 M in MeCN, 2.0 mL) wereadded followed by stirring at room temperature for 14 h. The reactionmixture was quenched by addition of excess 5% aqueous NaHCO₃. Theemulsion was diluted with 5% aqueous NaHCO₃ (30 mL), and the product wasextracted with ethyl acetate (3×50 mL). Extracts were washed with brine,dried over anhydrous Na₂SO₄, and evaporated to oil. Evaporation offractions gave the title compound as white solid foam in a yield of 1851mg (67.3%). ³¹P NMR (mixture of diastereomers, CD₃CN): δ 148.83, 148.23.

Example 45N⁶-Phenoxyacetyl-5′-O-(4,4′-dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[5-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3-oxapentyloxy]phosphinyl]-2′-deoxyadenosine(30b)

Solution of bis(N,N-diisopropylamino) chlorophosphite (700, 2.63 mmol)in anhydrous dichloromethane (5 mL) was added dropwise to a stirredsolution ofN⁶-phenoxyacetyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyadenosine, 10b,(1444 mg, 2.10 mmol) and N-ethyl-N,N-diisopropylamine (515 mg, 3.99mmol) in anhydrous dichloromethane (10 mL) at −20° C. The solution wasallowed to stir at room temperature for 2 h. To the obtained mixture,compound 7b (1097 mg, 2.52 mmol) and 1H-tetrazole (0.45 M in MeCN, 1.9mL) were added followed by stirring at room temperature for 14 h. Thereaction mixture was quenched by addition of excess 5% aqueous NaHCO₃.The emulsion was diluted with 5% aqueous NaHCO₃ (50 mL), and the productwas extracted with ethyl acetate (3×50 mL). Extracts were washed withbrine, dried over anhydrous Na₂SO₄, and evaporated to oil. Evaporationof fractions gave the title compound as white solid foam in a yield of1681 mg (63.9%). ³¹P NMR (mixture of diastereomers, CD₃CN): δ 147.94,147.52.

Example 46N²-Isobutyryl-5′-O-(4,4′-dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[5-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3-oxapentyloxy]phosphinyl]-2′-deoxyguanosine(31a)

Solution of bis(N,N-diisopropylamino) chlorophosphite (734 mg, 2.75mmol) in anhydrous dichloromethane (5 mL) was added dropwise to astirred solution ofN²-isobutyryl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyguanosine, 11a, (1407mg, 2.20 mmol) and N-ethyl-N,N-diisopropylamine (540 mg, 4.18 mmol) inanhydrous dichloromethane (10 mL) at −20° C. The solution was allowed tostir at room temperature for 2 h. To the obtained mixture, compound 7b(1149 mg, 2.64 mmol) and 1H-tetrazole (0.45 M in MeCN, 2.0 mL) wereadded followed by stirring at room temperature for 14 h. The reactionmixture was quenched by addition of excess 5% aqueous NaHCO₃. Theemulsion was diluted with 5% aqueous NaHCO₃ (30 mL), and the product wasextracted with ethyl acetate (3×50 mL). Extracts were washed with brine,dried over anhydrous Na₂SO₄, and evaporated to oil. Evaporation offractions gave the title compound as white solid foam in a yield of 1706mg (64.4%). ³¹P NMR (mixture of diastereomers, CD₃CN): δ 147.88, 147.43.

Example 47N²-Phenoxyacetyl-5′-O-(4,4′-dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[5-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3-oxapentyloxy]phosphinyl]-2′-deoxyguanosine(31b)

Solution of bis(N,N-diisopropylamino) chlorophosphite (750 mg, 2.81mmol) in anhydrous dichloromethane (5 mL) was added dropwise to astirred solution ofN²-phenoxyacetyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyguanosine, 11b,(1583 mg, 2.25 mmol) and N-ethyl-N,N-diisopropylamine (552 mg, 4.28mmol) in anhydrous dichloromethane (10 mL) at −20° C. The solution wasallowed to stir at room temperature for 2 h. To the obtained mixture,compound 7b (1176 mg, 2.70 mmol) and 1H-tetrazole (0.45 M in MeCN, 2.0mL) were added followed by stirring at room temperature for 14 h. Thereaction mixture was quenched by addition of excess 5% aqueous NaHCO₃.The emulsion was diluted with 5% aqueous NaHCO₃ (50 mL), and the productwas extracted with ethyl acetate (3×50 mL). Extracts were washed withbrine, dried over anhydrous Na₂SO₄, and evaporated to oil. Evaporationof fractions gave the title compound as white solid foam in a yield of1758 mg (61.6%). ³¹P NMR (mixture of diastereomers, CD₃CN): δ 147.80,147.39.

Example 481-O-(8-Hydroxy-3,6-dioxaoctyl)-3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranoside(7c)

A mixture of compound 5 (3.29 g, 10.0 mmol), anhydrous triethyleneglycol(12.0 g, 80 mmol), and pyridinium p-toluenesulfonate (2.26 g, 9.0 mmol)was heated at 80° C. for 18 h, then distributed between DCM (70 mL) anda 1:1 mixture of brine and 5% aqueous NaHCO₃ (100 mL). The organic phasewas separated, dried over Na₂SO₄, and evaporated in vacuo. The productwas isolated by column chromatography on silica gel eluted with a stepgradient from DCM to a mixture of MeOH and DCM (8:92). Evaporation ofthe relevant fractions gave the title compound as an off-white solid(2.32 g, 48.4%). ES MS: 480.2 (MH⁺). Calculated for C₁₈H₂₉NO₁₁.H⁺:480.2.

Example 495′-O-(4,4′-Dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[8-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6-dioxaoctyloxy]phosphinyl]thymidine(32)

Solution of bis(N,N-diisopropylamino) chlorophosphite (347 mg, 1.30mmol) in anhydrous dichloromethane (5 mL) was added dropwise to astirred solution of 5′-O-(4,4′-dimethoxytrityl)thymidine, 8, (566 mg,1.04 mmol) and N-ethyl-N,N-diisopropylamine (255 mg, 1.98 mmol) inanhydrous dichloromethane (5 mL) at −20° C. The solution was allowed tostir at room temperature for 2 h. To the obtained mixture,1-O-(8-hydroxy-3,6-dioxaoctyl)-3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranoside,7c, (598 mg, 1.25 mmol) and 1H-tetrazole (0.45 M in MeCN, 1.0 mL) wereadded followed by stirring at room temperature for 14 h. The reactionmixture was quenched by addition of excess 5% aqueous NaHCO₃. Theemulsion was diluted with 5% aqueous NaHCO₃ (50 mL), and the product wasextracted with ethyl acetate (3×50 mL). Extracts were washed with brine,dried over anhydrous Na₂SO₄, and evaporated to oil. The product wasisolated by column chromatography on silica gel eluted with a stepgradient starting from TEA—ethyl acetate—hexane (5:20:75) to a mixtureof TEA in ethyl acetate (5:95). Evaporation of fractions gave the titlecompound as white solid foam in a yield of 920 mg (76.7%). ³¹P NMR(mixture of diastereomers, CD₃CN): δ 147.95, 147.41.

Example 50N⁴-Benzoyl-5′-O-(4,4′-dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[8-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6-dioxaoctyloxy]phosphinyl]-2′-deoxycytidine(33a)

Solution of bis(N,N-diisopropylamino) chlorophosphite (357 mg, 1.34mmol) in anhydrous dichloromethane (5 mL) was added dropwise to astirred solution ofN⁴-benzoyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxycytidine, 9a, (678 mg,1.7 mmol) and N-ethyl-N,N-diisopropylamine (263 mg, 2.03 mmol) inanhydrous dichloromethane (5 mL) at −20° C. The solution was allowed tostir at room temperature for 2 h. To the obtained mixture, compound 7c(616 mg, 1.28 mmol) and 1H-tetrazole (0.45 M in MeCN, 1.0 mL) were addedfollowed by stirring at room temperature for 14 h. The reaction mixturewas quenched by addition of excess 5% aqueous NaHCO₃. The emulsion wasdiluted with 5% aqueous NaHCO₃ (50 mL), and the product was extractedwith ethyl acetate (3×50 mL). Extracts were washed with brine, driedover anhydrous Na₂SO₄, and evaporated to oil. The product was isolatedby column chromatography on silica gel eluted with a step gradientstarting from TEA—ethyl acetate—hexane (5:20:75) to a mixture of TEA inethyl acetate (5:95). Evaporation of fractions gave the title compoundas white solid foam in a yield of 980 mg (73.7%). ³¹P NMR (mixture ofdiastereomers, CD₃CN): δ 148.22, 147.62.

Example 51N⁴-Acetyl-5′-O-(4,4′-dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[8-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6-dioxaoctyloxy]phosphinyl]-2′-deoxycytidine(33c)

Solution of bis(N,N-diisopropylamino) chlorophosphite (333 mg, 1.25mmol) in anhydrous dichloromethane (5 mL) was added dropwise to astirred solution ofN⁴-acetyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxycytidine, 9c, (572 mg, 1.0mmol) and N-ethyl-N,N-diisopropylamine (245 mg, 1.90 mmol) in anhydrousdichloromethane (5 mL) at −20° C. The solution was allowed to stir atroom temperature for 2 h. To the obtained mixture, compound 7c (575 mg,1.20 mmol) and 1H-tetrazole (0.45 M in MeCN, 1.0 mL) were added followedby stirring at room temperature for 14 h. The reaction mixture wasquenched by addition of excess 5% aqueous NaHCO₃. The emulsion wasdiluted with 5% aqueous NaHCO₃ (30 mL), and the product was extractedwith ethyl acetate (3×50 mL). Extracts were washed with brine, driedover anhydrous Na₂SO₄, and evaporated to oil. The product was isolatedby column chromatography on silica gel eluted with a step gradientstarting from TEA—ethyl acetate—hexane (5:20:75) to a mixture of TEA inethyl acetate (5:95). Evaporation of fractions gave the title compoundas white solid foam in a yield of 829 mg (70.2%). ³¹P NMR (mixture ofdiastereomers, CD₃CN): δ 148.08, 147.72.

Example 52N⁶-Benzoyl-5′-O-(4,4′-dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[8-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6-dioxaoctyloxy]phosphinyl]-2′-deoxyadenosine(34a)

Solution of bis(N,N-diisopropylamino) chlorophosphite (340 mg, 1.28mmol) in anhydrous dichloromethane (5 mL) was added dropwise to astirred solution ofN⁶-benzoyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyadenosine, 10a, (671,1.02 mmol) and N-ethyl-N,N-diisopropylamine (250 mg, 1.94 mmol) inanhydrous dichloromethane (5 mL) at −20° C. The solution was allowed tostir at room temperature for 2 h. To the obtained mixture, compound 7c(587 mg, 1.22 mmol) and 1H-tetrazole (0.45 M in MeCN, 0.9 mL) were addedfollowed by stirring at room temperature for 14 h. The reaction mixturewas quenched by addition of excess 5% aqueous NaHCO₃. The emulsion wasdiluted with 5% aqueous NaHCO₃ (50 mL), and the product was extractedwith ethyl acetate (3×50 mL). Extracts were washed with brine, driedover anhydrous Na₂SO₄, and evaporated to oil. Evaporation of fractionsgave the title compound as white solid foam in a yield of 831 mg(64.3%). ³¹P NMR (mixture of diastereomers, CD₃CN): δ 148.19, 147.47.

Example 53N⁶-Phenoxyacetyl-5′-O-(4,4′-dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[8-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6-dioxaoctyloxy]phosphinyl]-2′-deoxyadenosine(34b)

Solution of bis(N,N-diisopropylamino) chlorophosphite (370, 1.39 mmol)in anhydrous dichloromethane (5 mL) was added dropwise to a stirredsolution ofN⁶-phenoxyacetyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyadenosine, 10b,(763 mg, 1.11 mmol) and N-ethyl-N,N-diisopropylamine (272 mg, 2.11 mmol)in anhydrous dichloromethane (5 mL) at −20° C. The solution was allowedto stir at room temperature for 2 h. To the obtained mixture, compound7c (639 mg, 1.33 mmol) and 1H-tetrazole (0.45 M in MeCN, 1.0 mL) wereadded followed by stirring at room temperature for 14 h. The reactionmixture was quenched by addition of excess 5% aqueous NaHCO₃. Theemulsion was diluted with 5% aqueous NaHCO₃ (40 mL), and the product wasextracted with ethyl acetate (3×50 mL). Extracts were washed with brine,dried over anhydrous Na₂SO₄, and evaporated to oil. Evaporation offractions gave the title compound as white solid foam in a yield of 872mg (60.6%). ³¹P NMR (mixture of diastereomers, CD₃CN): δ 148.51, 147.90.

Example 54N²-Isobutyryl-5′-O-(4,4′-dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[8-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6-dioxaoctyloxy]phosphinyl]-2′-deoxyguanosine(35a)

Solution of bis(N,N-diisopropylamino) chlorophosphite (320 mg, 1.20mmol) in anhydrous dichloromethane (5 mL) was added dropwise to astirred solution ofN²-isobutyryl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyguanosine, 11a, (614mg, 0.96 mmol) and N-ethyl-N,N-diisopropylamine (236 mg, 1.82 mmol) inanhydrous dichloromethane (5 mL) at −20° C. The solution was allowed tostir at room temperature for 2 h. To the obtained mixture, compound 7c(552 mg, 1.15 mmol) and 1H-tetrazole (0.45 M in MeCN, 0.9 mL) were addedfollowed by stirring at room temperature for 14 h. The reaction mixturewas quenched by addition of excess 5% aqueous NaHCO₃. The emulsion wasdiluted with 5% aqueous NaHCO₃ (40 mL), and the product was extractedwith ethyl acetate (3×50 mL). Extracts were washed with brine, driedover anhydrous Na₂SO₄, and evaporated to oil. Evaporation of fractionsgave the title compound as white solid foam in a yield of 784 mg(65.4%). ³¹P NMR (mixture of diastereomers, CD₃CN): δ 147.60, 147.11.

Example 55N²-Phenoxyacetyl-5′-O-(4,4′-dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[8-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6-dioxaoctyloxy]phosphinyl]-2′-deoxyguanosine(35b)

Solution of bis(N,N-diisopropylamino) chlorophosphite (330 mg, 1.24mmol) in anhydrous dichloromethane (5 mL) was added dropwise to astirred solution ofN²-phenoxyacetyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyguanosine, 11b,(697 mg, 0.99 mmol) and N-ethyl-N,N-diisopropylamine (243 mg, 1.88 mmol)in anhydrous dichloromethane (10 mL) at −20° C. The solution was allowedto stir at room temperature for 2 h. To the obtained mixture, compound7c (570 mg, 1.19 mmol) and 1H-tetrazole (0.45 M in MeCN, 0.9 mL) wereadded followed by stirring at room temperature for 14 h. The reactionmixture was quenched by addition of excess 5% aqueous NaHCO₃. Theemulsion was diluted with 5% aqueous NaHCO₃ (30 mL), and the product wasextracted with ethyl acetate (3×50 mL). Extracts were washed with brine,dried over anhydrous Na₂SO₄, and evaporated to oil. Evaporation offractions gave the title compound as white solid foam in a yield of 803mg (61.8%). ³¹P NMR (mixture of diastereomers, CD₃CN): δ 148.55, 147.73.

Example 561-O-(11-Hydroxy-3,6,9-trioxaundecyl)-3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranoside(7d)

A mixture of compound 5 (7.25 g, 22.0 mmol), anhydroustetraethyleneglycol (34.2 g, 176 mmol), and pyridiniump-toluenesulfonate (4.98 g, 19.8 mmol) was heated at 80° C. for 18 h,then distributed between DCM (50 mL) and a 1:1 mixture of brine and 5%aqueous NaHCO₃ (100 mL). The organic phase was separated, dried overNa₂SO₄, and evaporated in vacuo. The product was isolated by columnchromatography on silica gel eluted with a step gradient from DCM to amixture of MeOH and DCM (8:92). Evaporation of the relevant fractionsgave the title compound as an off-white solid (4.92 g, 42.7%). ES MS:524.4 (MH⁺). Calculated for C₁₈H₂₉NO₁₁.H⁺: 524.2.

Example 575′-O-(4,4′-Dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[11-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6,9-trioxaundecyloxy]phosphinyl]thymidine(36)

Solution of bis(N,N-diisopropylamino) chlorophosphite (370 mg, 1.39mmol) in anhydrous dichloromethane (5 mL) was added dropwise to astirred solution of 5′-O-(4,4′-dimethoxytrityl)thymidine, 8, (604 mg,1.11 mmol) and N-ethyl-N,N-diisopropylamine (272 mg, 2.11 mmol) inanhydrous dichloromethane (5 mL) at −20° C. The solution was allowed tostir at room temperature for 2 h. To the obtained mixture,1-O-(11-hydroxy-3,6,9-trioxaundecyl)-3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranoside,7d, (697 mg, 1.33 mmol) and 1H-tetrazole (0.45 M in MeCN, 1.0 mL) wereadded followed by stirring at room temperature for 14 h. The reactionmixture was quenched by addition of excess 5% aqueous NaHCO₃. Theemulsion was diluted with 5% aqueous NaHCO₃ (30 mL), and the product wasextracted with ethyl acetate (3×50 mL). Extracts were washed with brine,dried over anhydrous Na₂SO₄, and evaporated to oil. The product wasisolated by column chromatography on silica gel eluted with a stepgradient starting from TEA—ethyl acetate—hexane (5:20:75) to a mixtureof TEA in ethyl acetate (5:95). Evaporation of fractions gave the titlecompound as a viscous oil in a yield of 1099 mg (82.7%). ³¹P NMR(mixture of diastereomers, CD₃CN; see FIG. 10): δ 152.37, 152.3.

Example 58N⁴-Benzoyl-5′-O-(4,4′-dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[11-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6,9-trioxaundecyloxy]phosphinyl]-2′-deoxycytidine(37a)

Solution of bis(N,N-diisopropylamino) chlorophosphite (313 mg, 1.18mmol) in anhydrous dichloromethane (5 mL) was added dropwise to astirred solution ofN⁴-benzoyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxycytidine, 9a, (596 mg,0.94 mmol) and N-ethyl-N,N-diisopropylamine (231 mg, 1.79 mmol) inanhydrous dichloromethane (5 mL) at −20° C. The solution was allowed tostir at room temperature for 2 h. To the obtained mixture, compound 7d(590 mg, 1.13 mmol) and 1H-tetrazole (0.45 M in MeCN, 1.0 mL) were addedfollowed by stirring at room temperature for 14 h. The reaction mixturewas quenched by addition of excess 5% aqueous NaHCO₃. The emulsion wasdiluted with 5% aqueous NaHCO₃ (30 mL), and the product was extractedwith ethyl acetate (3×50 mL). Extracts were washed with brine, driedover anhydrous Na₂SO₄, and evaporated to oil. The product was isolatedby column chromatography on silica gel eluted with a step gradientstarting from TEA—ethyl acetate—hexane (5:20:75) to a mixture of TEA inethyl acetate (5:95). Evaporation of fractions gave the title compoundas a viscous oil in a yield of 940 mg (77.7%). ³¹P NMR (mixture ofdiastereomers, CD₃CN): δ 150.06, 149.74.

Example 59N⁴-Phenoxyacetyl-5′-O-(4,4′-dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[11-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6,9-trioxaundecyloxy]phosphinyl]-2′-deoxycytidine(37b)

Solution of bis(N,N-diisopropylamino) chlorophosphite (323 mg, 1.21mmol) in anhydrous dichloromethane (5 mL) was added dropwise to astirred solution ofN⁴⁻phenoxyacetyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxycytidine, 9b, (644mg, 0.97 mmol) and N-ethyl-N,N-diisopropylamine (238 mg, 1.84 mmol) inanhydrous dichloromethane (5 mL) at −20° C. The solution was allowed tostir at room temperature for 2 h. To the obtained mixture, compound 7d(609 mg, 1.16 mmol) and 1H-tetrazole (0.45 M in MeCN, 0.9 mL) were addedfollowed by stirring at room temperature for 14 h. The reaction mixturewas quenched by addition of excess 5% aqueous NaHCO₃. The emulsion wasdiluted with 5% aqueous NaHCO₃ (30 mL), and the product was extractedwith ethyl acetate (3×50 mL). Extracts were washed with brine, driedover anhydrous Na₂SO₄, and evaporated to oil. The product was isolatedby column chromatography on silica gel eluted with a step gradientstarting from TEA—ethyl acetate-hexane (5:20:75) to a mixture of TEA inethyl acetate (5:95). Evaporation of fractions gave the title compoundas a viscous oil in a yield of 1014 mg (79.4%). ³¹P NMR (mixture ofdiastereomers, CD₃CN): δ 151.67, 151.24.

Example 60N⁴-Acetyl-5′-O-(4,4′-dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[11-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6,9-trioxaundecyloxy]phosphinyl]-2′-deoxycytidine(37c)

Solution of bis(N,N-diisopropylamino) chlorophosphite (340 mg, 1.28mmol) in anhydrous dichloromethane (5 mL) was added dropwise to astirred solution ofN⁴-acetyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxycytidine, 9c, (583 mg,1.02 mmol) and N-ethyl-N,N-diisopropylamine (250 mg, 1.94 mmol) inanhydrous dichloromethane (5 mL) at −20° C. The solution was allowed tostir at room temperature for 2 h. To the obtained mixture, compound 7d(641 mg, 1.22 mmol) and 1H-tetrazole (0.45 M in MeCN, 1.0 mL) were addedfollowed by stirring at room temperature for 14 h. The reaction mixturewas quenched by addition of excess 5% aqueous NaHCO₃. The emulsion wasdiluted with 5% aqueous NaHCO₃ (30 mL), and the product was extractedwith ethyl acetate (3×50 mL). Extracts were washed with brine, driedover anhydrous Na₂SO₄, and evaporated to oil. The product was isolatedby column chromatography on silica gel eluted with a step gradientstarting from TEA—ethyl acetate—hexane (5:20:75) to a mixture of TEA inethyl acetate (5:95). Evaporation of fractions gave the title compoundas viscous oil in a yield of 998 mg (79.9%). ³¹P NMR (mixture ofdiastereomers, CD₃CN): δ 149.82, 149.19.

Example 61N⁶-Benzoyl-5′-O-(4,4′-dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[11-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6,9-trioxaundecyloxy]phosphinyl]-2′-deoxyadenosine(38a)

Solution of bis(N,N-diisopropylamino) chlorophosphite (327 mg, 1.23mmol) in anhydrous dichloromethane (5 mL) was added dropwise to astirred solution ofN⁶-benzoyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyadenosine, 10a, (645,0.98 mmol) and N-ethyl-N,N-diisopropylamine (240 mg, 1.86 mmol) inanhydrous dichloromethane (5 mL) at −20° C. The solution was allowed tostir at room temperature for 2 h. To the obtained mixture, compound 7d(616 mg, 1.18 mmol) and 1H-tetrazole (0.45 M in MeCN, 0.9 mL) were addedfollowed by stirring at room temperature for 14 h. The reaction mixturewas quenched by addition of excess 5% aqueous NaHCO₃. The emulsion wasdiluted with 5% aqueous NaHCO₃ (50 mL), and the product was extractedwith ethyl acetate (3×50 mL). Extracts were washed with brine, driedover anhydrous Na₂SO₄, and evaporated to oil. Evaporation of fractionsgave the title compound as a viscous oil in a yield of 944 mg (73.5%).³¹P NMR (mixture of diastereomers, CD₃CN): δ 150.09, 149.36.

Example 62N⁶-Phenoxyacetyl-5′-O-(4,4′-dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[11-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6,9-trioxaundecyloxy]phosphinyl]-2′-deoxyadenosine(38b)

Solution of bis(N,N-diisopropylamino) chlorophosphite (333, 1.25 mmol)in anhydrous dichloromethane (5 mL) was added dropwise to a stirredsolution ofN⁶-phenoxyacetyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyadenosine, 10b,(688 mg, 1.0 mmol) and N-ethyl-N,N-diisopropylamine (245 mg, 1.90 mmol)in anhydrous dichloromethane (5 mL) at −20° C. The solution was allowedto stir at room temperature for 2 h. To the obtained mixture, compound7d (628 mg, 1.20 mmol) and 1H-tetrazole (0.45 M in MeCN, 0.9 mL) wereadded followed by stirring at room temperature for 14 h. The reactionmixture was quenched by addition of excess 5% aqueous NaHCO₃. Theemulsion was diluted with 5% aqueous NaHCO₃ (40 mL), and the product wasextracted with ethyl acetate (3×50 mL). Extracts were washed with brine,dried over anhydrous Na₂SO₄, and evaporated to oil. Evaporation offractions gave the title compound as a viscous oil in a yield of 913 mg(68.1%). ³¹P NMR (mixture of diastereomers, CD₃CN): δ 149.84, 149.23.

Example 63N⁶-(4-i-Propylphenoxyacetyl)-5′-O-(4,4′-dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[11-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6,9-trioxaundecyloxy]phosphinyl]-2′-deoxyadenosine(38c)

Solution of bis(N,N-diisopropylamino) chlorophosphite (340, 1.28 mmol)in anhydrous dichloromethane (5 mL) was added dropwise to a stirredsolution of N⁶-(4-i-propylphenoxyacetyl)-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyadenosine, 10c, (744 mg,1.02 mmol) and N-ethyl-N,N-diisopropylamine (250 mg, 1.94 mmol) inanhydrous dichloromethane (5 mL) at −20° C. The solution was allowed tostir at room temperature for 2 h. To the obtained mixture, compound 7d(641 mg, 1.22 mmol) and 1H-tetrazole (0.45 M in MeCN, 1.0 mL) were addedfollowed by stirring at room temperature for 14 h. The reaction mixturewas quenched by addition of excess 5% aqueous NaHCO₃. The emulsion wasdiluted with 5% aqueous NaHCO₃ (40 mL), and the product was extractedwith ethyl acetate (3×50 mL). Extracts were washed with brine, driedover anhydrous Na₂SO₄, and evaporated to oil. Evaporation of fractionsgave the title compound as a viscous oil in a yield of 982 mg (69.6%).³¹P NMR (mixture of diastereomers, CD₃CN): δ 149.91, 149.42.

Example 64N⁶—(N,N-dimethylformamidino)-5′-O-(4,4′-dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[11-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6,9-trioxaundecyloxy]phosphinyl]-2′-deoxyadenosine(38d)

Solution of bis(N,N-diisopropylamino) chlorophosphite (320, 1.20 mmol)in anhydrous dichloromethane (5 mL) was added dropwise to a stirredsolution ofN⁶—(N,N-dimethylformamidino)-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyadenosine,10d, (584 mg, 0.96 mmol) and N-ethyl-N,N-diisopropylamine (236 mg, 1.82mmol) in anhydrous dichloromethane (5 mL) at −20° C. The solution wasallowed to stir at room temperature for 2 h. To the obtained mixture,compound 7d (603 mg, 1.15 mmol) and 1H-tetrazole (0.45 M in MeCN, 0.9mL) were added followed by stirring at room temperature for 14 h. Thereaction mixture was quenched by addition of excess 5% aqueous NaHCO₃.The emulsion was diluted with 5% aqueous NaHCO₃ (40 mL), and the productwas extracted with ethyl acetate (3×50 mL). Extracts were washed withbrine, dried over anhydrous Na₂SO₄, and evaporated to oil. Evaporationof fractions gave the title compound as a viscous oil in a yield of 798mg (65.9%). ³¹P NMR (mixture of diastereomers, CD₃CN): δ 151.19, 150.55.

Example 65N²-Isobutyryl-5′-O-(4,4′-dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[11-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6,9-trioxaundecyloxy]phosphinyl]-2′-deoxyguanosine(39a)

Solution of bis(N,N-diisopropylamino) chlorophosphite (343 mg, 1.29mmol) in anhydrous dichloromethane (5 mL) was added dropwise to astirred solution ofN²-isobutyryl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyguanosine, 11a, (659mg, 1.03 mmol) and N-ethyl-N,N-diisopropylamine (253 mg, 1.96 mmol) inanhydrous dichloromethane (5 mL) at −20° C. The solution was allowed tostir at room temperature for 2 h. To the obtained mixture, compound 7d(647 mg, 1.24 mmol) and 1H-tetrazole (0.45 M in MeCN, 0.9 mL) were addedfollowed by stirring at room temperature for 14 h. The reaction mixturewas quenched by addition of excess 5% aqueous NaHCO₃. The emulsion wasdiluted with 5% aqueous NaHCO₃ (40 mL), and the product was extractedwith ethyl acetate (3×50 mL). Extracts were washed with brine, driedover anhydrous Na₂SO₄, and evaporated to oil. Evaporation of fractionsgave the title compound as a viscous oil in a yield of 900 mg (67.6%).³¹P NMR (mixture of diastereomers, CD₃CN): δ 152.01, 151.92.

Example 66N²-Phenoxyacetyl-5′-O-(4,4′-dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[11-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6,9-trioxaundecyloxy]phosphinyl]-2′-deoxyguanosine(39b)

Solution of bis(N,N-diisopropylamino) chlorophosphite (340 mg, 1.28mmol) in anhydrous dichloromethane (5 mL) was added dropwise to astirred solution ofN²-phenoxyacetyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyguanosine, 11b,(718 mg, 1.02 mmol) and N-ethyl-N,N-diisopropylamine (250 mg, 1.94 mmol)in anhydrous dichloromethane (10 mL) at −20° C. The solution was allowedto stir at room temperature for 2 h. To the obtained mixture, compound7d (641 mg, 1.22 mmol) and 1H-tetrazole (0.45 M in MeCN, 1.0 mL) wereadded followed by stirring at room temperature for 14 h. The reactionmixture was quenched by addition of excess 5% aqueous NaHCO₃. Theemulsion was diluted with 5% aqueous NaHCO₃ (30 mL), and the product wasextracted with ethyl acetate (3×50 mL). Extracts were washed with brine,dried over anhydrous Na₂SO₄, and evaporated to oil. Evaporation offractions gave the title compound as a viscous oil in a yield of 944 mg(68.2%). ³¹P NMR (mixture of diastereomers, CD₃CN): δ 151.95, 151.36.

Example 67N²-(4-i-Propylphenoxyacetyl)-5′-O-(4,4′-dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[11-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6,9-trioxaundecyl]-3-oxapentyloxy]phosphinyl]-2′-deoxyguanosine(39c)

Solution of bis(N,N-diisopropylamino) chlorophosphite (333 mg, 1.25mmol) in anhydrous dichloromethane (5 mL) was added dropwise to astirred solution of N²-(4-i-propylphenoxyacetyl)-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyguanosine, 11c, (746 mg, 1.0mmol) and N-ethyl-N,N-diisopropylamine (245 mg, 1.90 mmol) in anhydrousdichloromethane (10 mL) at −20° C. The solution was allowed to stir atroom temperature for 2 h. To the obtained mixture, compound 7d (628 mg,1.20 mmol) and 1H-tetrazole (0.45 M in MeCN, 0.9 mL) were added followedby stirring at room temperature for 14 h. The reaction mixture wasquenched by addition of excess 5% aqueous NaHCO₃. The emulsion wasdiluted with 5% aqueous NaHCO₃ (30 mL), and the product was extractedwith ethyl acetate (3×50 mL). Extracts were washed with brine, driedover anhydrous Na₂SO₄, and evaporated to oil. Evaporation of fractionsgave the title compound as a viscous oil in a yield of 860 mg (61.5%).³¹P NMR (mixture of diastereomers, CD₃CN): δ 150.29, 149.95.

Example 68N²—(N,N-Dimethylformamidino)-5′-O-(4,4′-dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[11-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6,9-trioxaundecyloxy]phosphinyl]-2′-deoxyguanosine(39d)

Solution of bis(N,N-diisopropylamino) chlorophosphite (317 mg, 1.19mmol) in anhydrous dichloromethane (5 mL) was added dropwise to astirred solution ofN²—(N,N-dimethylformamidino)-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyguanosine,11d, (594 mg, 0.95 mmol) and N-ethyl-N,N-diisopropylamine (233 mg, 1.81mmol) in anhydrous dichloromethane (10 mL) at −20° C. The solution wasallowed to stir at room temperature for 2 h. To the obtained mixture,compound 7d (597 mg, 1.14 mmol) and 1H-tetrazole (0.45 M in MeCN, 0.9mL) were added followed by stirring at room temperature for 14 h. Thereaction mixture was quenched by addition of excess 5% aqueous NaHCO₃.The emulsion was diluted with 5% aqueous NaHCO₃ (30 mL), and the productwas extracted with ethyl acetate (3×50 mL). Extracts were washed withbrine, dried over anhydrous Na₂SO₄, and evaporated to oil. Evaporationof fractions gave the title compound as a viscous oil in a yield of 808mg (66.6%). ³¹P NMR (mixture of diastereomers, CD₃CN): δ 150.03, 149.72.

Example 691-O-(14-Hydroxy-3,6,9,12-tetraoxatetradecyl)-3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranoside(7e)

A mixture of compound 5 (1.64 g, 5.0 mmol), anhydrouspentaethyleneglycol (7.15 g, 30.0 mol), and pyridiniump-toluenesulfonate (1.13 g, 4.5 mmol) was heated at 80° C. for 18 h,then distributed between DCM (50 mL) and a 1:1 mixture of brine and 5%aqueous NaHCO₃ (25 mL). The organic phase was separated, dried overNa₂SO₄, and evaporated in vacuo. The product was isolated by columnchromatography on silica gel eluted with a step gradient from DCM to amixture of MeOH and DCM (8:92). Evaporation of the relevant fractionsgave the title compound as an off-white solid (1.08 g, 38.1%). ES MS:568.0 (MH⁺). Calculated for C₁₈H₂₉NO₁₁.H⁺: 568.2.

Example 705′-O-(4,4′-Dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[14-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6,9,12-tetraoxatetradecyloxy]phosphinyl]thymidine(40)

Solution of bis(N,N-diisopropylamino) chlorophosphite (213 mg, 0.8 mmol)in anhydrous dichloromethane (3 mL) was added dropwise to a stirredsolution of 5′-O-(4,4′-dimethoxytrityl)thymidine, 8, (348 mg, 0.64 mmol)and N-ethyl-N,N-diisopropylamine (157 mg, 1.22 mmol) in anhydrousdichloromethane (4 mL) at −20° C. The solution was allowed to stir atroom temperature for 2 h. To the obtained mixture,1-O-(14-hydroxy-3,6,9,12-tetraoxatetradecyl)-3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranoside,7e, (436 mg, 0.77 mmol) and 1H-tetrazole (0.45 M in MeCN, 0.6 mL) wereadded followed by stirring at room temperature for 14 h. The reactionmixture was quenched by addition of excess 5% aqueous NaHCO₃. Theemulsion was diluted with 5% aqueous NaHCO₃ (10 mL), and the product wasextracted with ethyl acetate (3×50 mL). Extracts were washed with brine,dried over anhydrous Na₂SO₄, and evaporated to oil. The product wasisolated by column chromatography on silica gel eluted with a stepgradient starting from TEA—ethyl acetate—hexane (5:20:75) to a mixtureof TEA in ethyl acetate (5:95). Evaporation of fractions gave the titlecompound as a viscous oil in a yield of 617 mg (77.7%). ³¹P NMR (mixtureof diastereomers, CD₃CN): δ 149.99, 149.66.

Example 71N⁴-Benzoyl-5′-O-(4,4′-dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[14-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6,9,12-tetraoxatetradecyloxy]phosphinyl]-2′-deoxycytidine(41a)

Solution of bis(N,N-diisopropylamino) chlorophosphite (240 mg, 0.90mmol) in anhydrous dichloromethane (3 mL) was added dropwise to astirred solution ofN⁴-benzoyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxycytidine, 9a, (456 mg,0.72 mmol) and N-ethyl-N,N-diisopropylamine (177 mg, 1.37 mmol) inanhydrous dichloromethane (4 mL) at −20° C. The solution was allowed tostir at room temperature for 2 h. To the obtained mixture, compound 7e(490 mg, 0.86 mmol) and 1H-tetrazole (0.45 M in MeCN, 0.7 mL) were addedfollowed by stirring at room temperature for 14 h. The reaction mixturewas quenched by addition of excess 5% aqueous NaHCO₃. The emulsion wasdiluted with 5% aqueous NaHCO₃ (10 mL), and the product was extractedwith ethyl acetate (3×50 mL). Extracts were washed with brine, driedover anhydrous Na₂SO₄, and evaporated to oil. The product was isolatedby column chromatography on silica gel eluted with a step gradientstarting from TEA—ethyl acetate—hexane (5:20:75) to a mixture of TEA inethyl acetate (5:95). Evaporation of fractions gave the title compoundas a viscous oil in a yield of 692 mg (72.2%). ³¹P NMR (mixture ofdiastereomers, CD₃CN): δ 148.17, 147.63.

Example 72N⁴-Acetyl-5′-O-(4,4′-dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[14-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6,9,12-tetraoxatetradecyloxy]phosphinyl]-2′-deoxycytidine(41c)

Solution of bis(N,N-diisopropylamino) chlorophosphite (220 mg, 0.83mmol) in anhydrous dichloromethane (3 mL) was added dropwise to astirred solution ofN⁴-acetyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxycytidine, 9c, (377 mg,0.66 mmol) and N-ethyl-N,N-diisopropylamine (157 mg, 1.22 mmol) inanhydrous dichloromethane (4 mL) at −20° C. The solution was allowed tostir at room temperature for 2 h. To the obtained mixture, compound 7e(450 mg, 0.79 mmol) and 1H-tetrazole (0.45 M in MeCN, 0.6 mL) were addedfollowed by stirring at room temperature for 14 h. The reaction mixturewas quenched by addition of excess 5% aqueous NaHCO₃. The emulsion wasdiluted with 5% aqueous NaHCO₃ (10 mL), and the product was extractedwith ethyl acetate (3×50 mL). Extracts were washed with brine, driedover anhydrous Na₂SO₄, and evaporated to oil. The product was isolatedby column chromatography on silica gel eluted with a step gradientstarting from TEA—ethyl acetate—hexane (5:20:75) to a mixture of TEA inethyl acetate (5:95). Evaporation of fractions gave the title compoundas a viscous oil in a yield of 590 mg (70.5%). ³¹P NMR (mixture ofdiastereomers, CD₃CN): δ 147.88, 147.21.

Example 73N⁶-Benzoyl-5′-O-(4,4′-dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[14-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6,9,12-tetraoxatetradecyloxy]phosphinyl]-2′-deoxyadenosine(42a)

Solution of bis(N,N-diisopropylamino) chlorophosphite (200 mg, 0.75mmol) in anhydrous dichloromethane (3 mL) was added dropwise to astirred solution ofN⁶-benzoyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyadenosine, 10a, (395,0.60 mmol) and N-ethyl-N,N-diisopropylamine (147 mg, 1.14 mmol) inanhydrous dichloromethane (4 mL) at −20° C. The solution was allowed tostir at room temperature for 2 h. To the obtained mixture, compound 7e(409 mg, 0.72 mmol) and 1H-tetrazole (0.45 M in MeCN, 0.6 mL) were addedfollowed by stirring at room temperature for 14 h. The reaction mixturewas quenched by addition of excess 5% aqueous NaHCO₃. The emulsion wasdiluted with 5% aqueous NaHCO₃ (10 mL), and the product was extractedwith ethyl acetate (3×50 mL). Extracts were washed with brine, driedover anhydrous Na₂SO₄, and evaporated to oil. Evaporation of fractionsgave the title compound as a viscous oil in a yield of 563 mg (69.3%).³¹P NMR (mixture of diastereomers, CD₃CN): δ 147.82, 147.28.

Example 74N⁶-Phenoxyacetyl-5′-O-(4,4′-dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[14-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6,9,12-tetraoxatetradecyloxy]phosphinyl]-2′-deoxyadenosine(42b)

Solution of bis(N,N-diisopropylamino) chlorophosphite (230, 0.86 mmol)in anhydrous dichloromethane (3 mL) was added dropwise to a stirredsolution ofN⁶-phenoxyacetyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyadenosine, 10b,(475 mg, 0.69 mmol) and N-ethyl-N,N-diisopropylamine (169 mg, 1.31 mmol)in anhydrous dichloromethane (4 mL) at −20° C. The solution was allowedto stir at room temperature for 2 h. To the obtained mixture, compound7e (470 mg, 0.83 mmol) and 1H-tetrazole (0.45 M in MeCN, 0.7 mL) wereadded followed by stirring at room temperature for 14 h. The reactionmixture was quenched by addition of excess 5% aqueous NaHCO₃. Theemulsion was diluted with 5% aqueous NaHCO₃ (10 mL), and the product wasextracted with ethyl acetate (3×50 mL). Extracts were washed with brine,dried over anhydrous Na₂SO₄, and evaporated to oil. Evaporation offractions gave the title compound as a viscous oil in a yield of 610 mg(63.9%). ³¹P NMR (mixture of diastereomers, CD₃CN): δ 148.17, 147.52.

Example 75N²-Isobutyryl-5′-O-(4,4′-dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[14-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6,9,12-tetraoxatetradecyloxy]phosphinyl]-2′-deoxyguanosine(43a)

Solution of bis(N,N-diisopropylamino) chlorophosphite (203 mg, 0.76mmol) in anhydrous dichloromethane (3 mL) was added dropwise to astirred solution ofN²-isobutyryl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyguanosine, 11a, (390mg, 0.61 mmol) and N-ethyl-N,N-diisopropylamine (150 mg, 1.16 mmol) inanhydrous dichloromethane (4 mL) at −20° C. The solution was allowed tostir at room temperature for 2 h. To the obtained mixture, compound 7e(415 mg, 0.73 mmol) and 1H-tetrazole (0.45 M in MeCN, 0.6 mL) were addedfollowed by stirring at room temperature for 14 h. The reaction mixturewas quenched by addition of excess 5% aqueous NaHCO₃. The emulsion wasdiluted with 5% aqueous NaHCO₃ (10 mL), and the product was extractedwith ethyl acetate (3×50 mL). Extracts were washed with brine, driedover anhydrous Na₂SO₄, and evaporated to oil. Evaporation of fractionsgave the title compound as a viscous oil in a yield of 543 mg (66.6%).³¹P NMR (mixture of diastereomers, CD₃CN): δ 147.83, 147.24.

Example 76N²-Phenoxyacetyl-5′-O-(4,4′-dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[14-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6,9,12-tetraoxatetradecyl]phosphino]-2′-deoxyguanosine(43b)

Solution of bis(N,N-diisopropylamino) chlorophosphite (217 mg, 0.81mmol) in anhydrous dichloromethane (3 mL) was added dropwise to astirred solution ofN²-phenoxyacetyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxyguanosine, 11b,(457 mg, 0.65 mmol) and N-ethyl-N,N-diisopropylamine (160 mg, 1.24 mmol)in anhydrous dichloromethane (4 mL) at −20° C. The solution was allowedto stir at room temperature for 2 h. To the obtained mixture, compound7e (443 mg, 0.78 mmol) and 1H-tetrazole (0.45 M in MeCN, 0.6 mL) wereadded followed by stirring at room temperature for 14 h. The reactionmixture was quenched by addition of excess 5% aqueous NaHCO₃. Theemulsion was diluted with 5% aqueous NaHCO₃ (10 mL), and the product wasextracted with ethyl acetate (3×50 mL). Extracts were washed with brine,dried over anhydrous Na₂SO₄, and evaporated to oil. Evaporation offractions gave the title compound as a viscous oil in a yield of 565 mg(62.1%). ³¹P NMR (mixture of diastereomers, CD₃CN): δ 148.29, 147.47.

Example 775′-O-(4,4′-Dimethoxytrityl)-2′-O-methyl-3′-O—[(N,N-diisopropylamino)[11-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6,9-trioxaundecyloxy]phosphinyl]-5-methyluridine(52)

Solution of bis(N,N-diisopropylamino) chlorophosphite (313 mg, 1.18mmol) in anhydrous dichloromethane (3 mL) was added dropwise to astirred solution of5′-O-(4,4′-dimethoxytrityl)-3′-O-methyl-5-methyluridine, 44, (540 mg,0.94 mmol) and N-ethyl-N,N-diisopropylamine (231 mg, 1.79 mmol) inanhydrous dichloromethane (4 mL) at −20° C. The solution was allowed tostir at room temperature for 2 h. To the obtained mixture,1-O-(11-hydroxy-3,6,9-trioxaundecyl)-3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranoside,7d, (591 mg, 1.13 mmol) and 1H-tetrazole (0.45 M in MeCN, 0.9 mL) wereadded followed by stirring at room temperature for 14 h. The reactionmixture was quenched by addition of excess 5% aqueous NaHCO₃. Theemulsion was diluted with 5% aqueous NaHCO₃ (10 mL), and the product wasextracted with ethyl acetate (3×50 mL). Extracts were washed with brine,dried over anhydrous Na₂SO₄, and evaporated to oil. The product wasisolated by column chromatography on silica gel eluted with a stepgradient starting from TEA—ethyl acetate—hexane (5:20:75) to a mixtureof TEA in ethyl acetate (5:95). Evaporation of fractions gave the titlecompound as a viscous oil in a yield of 977 mg (84.7%). ³¹P NMR (mixtureof diastereomers, CD₃CN): δ 149.43, 148.62.

Example 78N⁴-Benzoyl-5′-O-(4,4′-dimethoxytrityl)-2′-O-methyl-3′-O—[(N,N-diisopropylamino)[11-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6,9-trioxaundecyloxy]phosphinyl]cytidine(53a)

Solution of bis(N,N-diisopropylamino) chlorophosphite (323 mg, 1.21mmol) in anhydrous dichloromethane (3 mL) was added dropwise to astirred solution ofN⁴-benzoyl-5′-O-(4,4′-dimethoxytrityl)-2′-O-methylcytidine, 45a, (646mg, 0.97 mmol) and N-ethyl-N,N-diisopropylamine (238 mg, 1.84 mmol) inanhydrous dichloromethane (4 mL) at −20° C. The solution was allowed tostir at room temperature for 2 h. To the obtained mixture, compound 7d(609 mg, 1.16 mmol) and 1H-tetrazole (0.45 M in MeCN, 0.9 mL) were addedfollowed by stirring at room temperature for 14 h. The reaction mixturewas quenched by addition of excess 5% aqueous NaHCO₃. The emulsion wasdiluted with 5% aqueous NaHCO₃ (10 mL), and the product was extractedwith ethyl acetate (3×50 mL). Extracts were washed with brine, driedover anhydrous Na₂SO₄, and evaporated to oil. The product was isolatedby column chromatography on silica gel eluted with a step gradientstarting from TEA—ethyl acetate—hexane (5:20:75) to a mixture of TEA inethyl acetate (5:95). Evaporation of fractions gave the title compoundas a viscous oil in a yield of 1038 mg (81.2%). ³¹P NMR (mixture ofdiastereomers, CD₃CN): δ 149.17, 148.75.

Example 79N⁴-Acetyl-5′-O-(4,4′-dimethoxytrityl)-2′-O-methyl-3′-O—[(N,N-diisopropylamino)[11-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6,9-trioxaundecyl]cytidine(53c)

Solution of bis(N,N-diisopropylamino) chlorophosphite (340 mg, 1.28mmol) in anhydrous dichloromethane (3 mL) was added dropwise to astirred solution ofN⁴-acetyl-5′-O-(4,4′-dimethoxytrityl)-2′-O-methylcytidine, 45c, (616 mg,1.02 mmol) and N-ethyl-N,N-diisopropylamine (250 mg, 1.94 mmol) inanhydrous dichloromethane (4 mL) at −20° C. The solution was allowed tostir at room temperature for 2 h. To the obtained mixture, compound 7d(641 mg, 1.22 mmol) and 1H-tetrazole (0.45 M in MeCN, 1.0 mL) were addedfollowed by stirring at room temperature for 14 h. The reaction mixturewas quenched by addition of excess 5% aqueous NaHCO₃. The emulsion wasdiluted with 5% aqueous NaHCO₃ (10 mL), and the product was extractedwith ethyl acetate (3×50 mL). Extracts were washed with brine, driedover anhydrous Na₂SO₄, and evaporated to oil. The product was isolatedby column chromatography on silica gel eluted with a step gradientstarting from TEA—ethyl acetate—hexane (5:20:75) to a mixture of TEA inethyl acetate (5:95). Evaporation of fractions gave the title compoundas a viscous oil in a yield of 1061 mg (82.8%). ³¹P NMR (mixture ofdiastereomers, CD₃CN): δ 148.98, 148.43.

Example 80N⁶-Benzoyl-5′-O-(4,4′-dimethoxytrityl)-2′-O-methyl-3′-O—[(N,N-diisopropylamino)[11-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6,9-trioxaundecyl]adenosine(54a)

Solution of bis(N,N-diisopropylamino) chlorophosphite (330 mg, 1.24mmol) in anhydrous dichloromethane (3 mL) was added dropwise to astirred solution ofN⁶-benzoyl-5′-O-(4,4′-dimethoxytrityl)-2′-O-methyladenosine, 46a, (681mg, 0.99 mmol) and N-ethyl-N,N-diisopropylamine (243 mg, 1.88 mmol) inanhydrous dichloromethane (4 mL) at −20° C. The solution was allowed tostir at room temperature for 2 h. To the obtained mixture, compound 7d(622 mg, 1.19 mmol) and 1H-tetrazole (0.45 M in MeCN, 0.9 mL) were addedfollowed by stirring at room temperature for 14 h. The reaction mixturewas quenched by addition of excess 5% aqueous NaHCO₃. The emulsion wasdiluted with 5% aqueous NaHCO₃ (10 mL), and the product was extractedwith ethyl acetate (3×50 mL). Extracts were washed with brine, driedover anhydrous Na₂SO₄, and evaporated to oil. Evaporation of fractionsgave the title compound as a viscous oil in a yield of 1039 mg (78.3%).³¹P NMR (mixture of diastereomers, CD₃CN): δ 148.92, 148.37.

Example 81N⁶-Phenoxyacetyl-5′-O-(4,4′-dimethoxytrityl)-2′-O-methyl-3′-O—[(N,N-diisopropylamino)[11-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6,9-trioxaundecyloxy]phosphinyl]adenosine(54b)

Solution of bis(N,N-diisopropylamino) chlorophosphite (343, 1.29 mmol)in anhydrous dichloromethane (3 mL) was added dropwise to a stirredsolution ofN⁶-phenoxyacetyl-5′-O-(4,4′-dimethoxytrityl)-2′-O-methyladenosine, 46b,(739 mg, 1.03 mmol) and N-ethyl-N,N-diisopropylamine (253 mg, 1.96 mmol)in anhydrous dichloromethane (4 mL) at −20° C. The solution was allowedto stir at room temperature for 2 h. To the obtained mixture, compound7d (647 mg, 1.24 mmol) and 1H-tetrazole (0.45 M in MeCN, 1.0 mL) wereadded followed by stirring at room temperature for 14 h. The reactionmixture was quenched by addition of excess 5% aqueous NaHCO₃. Theemulsion was diluted with 5% aqueous NaHCO₃ (10 mL), and the product wasextracted with ethyl acetate (3×50 mL). Extracts were washed with brine,dried over anhydrous Na₂SO₄, and evaporated to oil. Evaporation offractions gave the title compound as a viscous oil in a yield of 1011 mg(71.6%). ³¹P NMR (mixture of diastereomers, CD₃CN): δ 147.92, 147.47.

Example 82N²-Isobutyryl-5′-O-(4,4′-dimethoxytrityl)-2′-O-methyl-3′-O—[(N,N-diisopropylamino)[11-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6,9-trioxaundecyloxy]phosphinyl]guanosine(55a)

Solution of bis(N,N-diisopropylamino) chlorophosphite (317 mg, 1.19mmol) in anhydrous dichloromethane (3 mL) was added dropwise to astirred solution ofN²-isobutyryl-5′-O-(4,4′-dimethoxytrityl)-2′-O-methylguanosine, 47a,(636 mg, 0.95 mmol) and N-ethyl-N,N-diisopropylamine (233 mg, 1.81 mmol)in anhydrous dichloromethane (4 mL) at −20° C. The solution was allowedto stir at room temperature for 2 h. To the obtained mixture, compound7d (597 mg, 1.14 mmol) and 1H-tetrazole (0.45 M in MeCN, 0.9 mL) wereadded followed by stirring at room temperature for 14 h. The reactionmixture was quenched by addition of excess 5% aqueous NaHCO₃. Theemulsion was diluted with 5% aqueous NaHCO₃ (10 mL), and the product wasextracted with ethyl acetate (3×50 mL). Extracts were washed with brine,dried over anhydrous Na₂SO₄, and evaporated to oil. Evaporation offractions gave the title compound as a viscous oil in a yield of 966 mg(76.9%). ³¹P NMR (mixture of diastereomers, CD₃CN): δ 147.90, 147.32.

Example 83N²-Phenoxyacetyl-5′-O-(4,4′-dimethoxytrityl)-2′-O-methyl-3′-O—[(N,N-diisopropylamino)[11-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6,9-trioxaundecyl]phosphino]guanosine (55b)

Solution of bis(N,N-diisopropylamino) chlorophosphite (337 mg, 1.26mmol) in anhydrous dichloromethane (3 mL) was added dropwise to astirred solution ofN²-phenoxyacetyl-5′-O-(4,4′-dimethoxytrityl)-2′-O-methylguanosine, 47b,(741 mg, 1.01 mmol) and N-ethyl-N,N-diisopropylamine (248 mg, 1.92 mmol)in anhydrous dichloromethane (4 mL) at −20° C. The solution was allowedto stir at room temperature for 2 h. To the obtained mixture, compound7d (635 mg, 1.21 mmol) and 1H-tetrazole (0.45 M in MeCN, 0.9 mL) wereadded followed by stirring at room temperature for 14 h. The reactionmixture was quenched by addition of excess 5% aqueous NaHCO₃. Theemulsion was diluted with 5% aqueous NaHCO₃ (10 mL), and the product wasextracted with ethyl acetate (3×50 mL). Extracts were washed with brine,dried over anhydrous Na₂SO₄, and evaporated to oil. Evaporation offractions gave the title compound as a viscous oil in a yield of 1011 mg(72.2%). ³¹P NMR (mixture of diastereomers, CD₃CN): δ 147.51, 146.96.

Example 845′-O-(4,4′-Dimethoxytrityl)-2′-fluoro-3′-O—[(N,N-diisopropylamino)[11-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6,9-trioxaundecyl]phosphino]-2′-deoxy-5-methyluridine (56)

Solution of bis(N,N-diisopropylamino) chlorophosphite (350 mg, 1.31mmol) in anhydrous dichloromethane (3 mL) was added dropwise to astirred solution of5′-O-(4,4′-dimethoxytrityl)-2′-fluoro-2′-deoxy-5-methyluridine, 48, (591mg, 1.05 mmol) and N-ethyl-N,N-diisopropylamine (258 mg, 2.00 mmol) inanhydrous dichloromethane (4 mL) at −20° C. The solution was allowed tostir at room temperature for 2 h. To the obtained mixture,1-O-(11-hydroxy-3,6,9-trioxaundecyl)-3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranoside,7d, (660 mg, 1.26 mmol) and 1H-tetrazole (0.45 M in MeCN, 1.0 mL) wereadded followed by stirring at room temperature for 14 h. The reactionmixture was quenched by addition of excess 5% aqueous NaHCO₃. Theemulsion was diluted with 5% aqueous NaHCO₃ (10 mL), and the product wasextracted with ethyl acetate (3×50 mL). Extracts were washed with brine,dried over anhydrous Na₂SO₄, and evaporated to oil. The product wasisolated by column chromatography on silica gel eluted with a stepgradient starting from TEA—ethyl acetate—hexane (5:20:75) to a mixtureof TEA in ethyl acetate (5:95). Evaporation of fractions gave the titlecompound as a viscous oil in a yield of 1060 mg (83.1%). ³¹P NMR(mixture of diastereomers, CD₃CN): δ 148.38, 147.87.

Example 85N⁴-Benzoyl-5′-O-(4,4′-dimethoxytrityl)-2′-fluoro-3′-O—[(N,N-diisopropylamino)[11-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6,9-trioxaundecyl]phosphino]-2′-deoxycytidine(57a)

Solution of bis(N,N-diisopropylamino) chlorophosphite (343 mg, 1.31mmol) in anhydrous dichloromethane (3 mL) was added dropwise to astirred solution ofN⁴-benzoyl-5′-O-(4,4′-dimethoxytrityl)-2′-fluoro-2′-deoxycytidine, 49a,(673 mg, 1.03 mmol) and N-ethyl-N,N-diisopropylamine (253 mg, 1.96 mmol)in anhydrous dichloromethane (4 mL) at −20° C. The solution was allowedto stir at room temperature for 2 h. To the obtained mixture, compound7d (647 mg, 1.24 mmol) and 1H-tetrazole (0.45 M in MeCN, 1.0 mL) wereadded followed by stirring at room temperature for 14 h. The reactionmixture was quenched by addition of excess 5% aqueous NaHCO₃. Theemulsion was diluted with 5% aqueous NaHCO₃ (10 mL), and the product wasextracted with ethyl acetate (3×50 mL). Extracts were washed with brine,dried over anhydrous Na₂SO₄, and evaporated to oil. The product wasisolated by column chromatography on silica gel eluted with a stepgradient starting from TEA—ethyl acetate—hexane (5:20:75) to a mixtureof TEA in ethyl acetate (5:95). Evaporation of fractions gave the titlecompound as a viscous oil in a yield of 1075 mg (79.9%). ³¹P NMR(mixture of diastereomers, CD₃CN): δ 148.52, 147.81.

Example 86N⁴-Acetyl-5′-O-(4,4′-dimethoxytrityl)-2′-fluoro-3′-O—[(N,N-diisopropylamino)[11-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6,9-trioxaundecyl]phosphino]-2′-deoxycytidine(57c)

Solution of bis(N,N-diisopropylamino) chlorophosphite (337 mg, 1.26mmol) in anhydrous dichloromethane (3 mL) was added dropwise to astirred solution ofN⁴-acetyl-5′-O-(4,4′-dimethoxytrityl)-2′-fluoro-2′-deoxycytidine, 49c,(598 mg, 1.01 mmol) and N-ethyl-N,N-diisopropylamine (248 mg, 1.92 mmol)in anhydrous dichloromethane (4 mL) at −20° C. The solution was allowedto stir at room temperature for 2 h. To the obtained mixture, compound7d (635 mg, 1.21 mmol) and 1H-tetrazole (0.45 M in MeCN, 1.0 mL) wereadded followed by stirring at room temperature for 14 h. The reactionmixture was quenched by addition of excess 5% aqueous NaHCO₃. Theemulsion was diluted with 5% aqueous NaHCO₃ (10 mL), and the product wasextracted with ethyl acetate (3×50 mL). Extracts were washed with brine,dried over anhydrous Na₂SO₄, and evaporated to oil. The product wasisolated by column chromatography on silica gel eluted with a stepgradient starting from TEA—ethyl acetate—hexane (5:20:75) to a mixtureof TEA in ethyl acetate (5:95). Evaporation of fractions gave the titlecompound as a viscous oil in a yield of 985 mg (78.4%). ³¹P NMR (mixtureof diastereomers, CD₃CN): δ 148.23, 147.69.

Example 87N⁶-Benzoyl-5′-O-(4,4′-dimethoxytrityl)-2′-fluoro-3′-O—[(N,N-diisopropylamino)[11-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6,9-trioxaundecyl]phosphino]-2′-deoxyadenosine(58a)

Solution of bis(N,N-diisopropylamino) chlorophosphite (347 mg, 1.30mmol) in anhydrous dichloromethane (3 mL) was added dropwise to astirred solution ofN⁶-benzoyl-5′-O-(4,4′-dimethoxytrityl)-2′-fluoro-2′-deoxyadenosine, 50a,(703 mg, 1.4 mmol) and N-ethyl-N,N-diisopropylamine (255 mg, 1.98 mmol)in anhydrous dichloromethane (4 mL) at −20° C. The solution was allowedto stir at room temperature for 2 h. To the obtained mixture, compound7d (653 mg, 1.25 mmol) and 1H-tetrazole (0.45 M in MeCN, 1.0 mL) wereadded followed by stirring at room temperature for 14 h. The reactionmixture was quenched by addition of excess 5% aqueous NaHCO₃. Theemulsion was diluted with 5% aqueous NaHCO₃ (10 mL), and the product wasextracted with ethyl acetate (3×50 mL). Extracts were washed with brine,dried over anhydrous Na₂SO₄, and evaporated to oil. Evaporation offractions gave the title compound as a viscous oil in a yield of 995 mg(72.0%). ³¹P NMR (mixture of diastereomers, CD₃CN): δ 148.23, 147.53.

Example 88N⁶-Phenoxyacetyl-5′-O-(4,4′-dimethoxytrityl)-2′-fluoro-3′-O—[(N,N-diisopropylamino)[11-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6,9-trioxaundecyl]phosphino]-2′-deoxyadenosine (58b)

Solution of bis(N,N-diisopropylamino) chlorophosphite (327, 1.23 mmol)in anhydrous dichloromethane (3 mL) was added dropwise to a stirredsolution ofN⁶-phenoxyacetyl-5′-O-(4,4′-dimethoxytrityl)-2′-fluoro-2′-deoxyadenosine,50b, (692 mg, 0.98 mmol) and N-ethyl-N,N-diisopropylamine (240 mg, 1.86mmol) in anhydrous dichloromethane (4 mL) at −20° C. The solution wasallowed to stir at room temperature for 2 h. To the obtained mixture,compound 7d (616 mg, 1.18 mmol) and 1H-tetrazole (0.45 M in MeCN, 0.9mL) were added followed by stirring at room temperature for 14 h. Thereaction mixture was quenched by addition of excess 5% aqueous NaHCO₃.The emulsion was diluted with 5% aqueous NaHCO₃ (10 mL), and the productwas extracted with ethyl acetate (3×50 mL). Extracts were washed withbrine, dried over anhydrous Na₂SO₄, and evaporated to oil. Evaporationof fractions gave the title compound as a viscous oil in a yield of 917mg (68.9%). ³¹P NMR (mixture of diastereomers, CD₃CN): δ 148.47, 147.88.

Example 89N²-Isobutyryl-5′-O-(4,4′-dimethoxytrityl)-2′-fluoro-3′-O—[(N,N-diisopropylamino)[11-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6,9-trioxaundecyl]phosphino]-2′-deoxyguanosine(59a)

Solution of bis(N,N-diisopropylamino) chlorophosphite (320 mg, 1.20mmol) in anhydrous dichloromethane (3 mL) was added dropwise to astirred solution ofN²-isobutyryl-5′-O-(4,4′-dimethoxytrityl)-2′-fluoro-2′-deoxyguanosine,51a, (631 mg, 0.96 mmol) and N-ethyl-N,N-diisopropylamine (236 mg, 1.82mmol) in anhydrous dichloromethane (4 mL) at −20° C. The solution wasallowed to stir at room temperature for 2 h. To the obtained mixture,compound 7d (603 mg, 1.15 mmol) and 1H-tetrazole (0.45 M in MeCN, 0.9mL) were added followed by stirring at room temperature for 14 h. Thereaction mixture was quenched by addition of excess 5% aqueous NaHCO₃.The emulsion was diluted with 5% aqueous NaHCO₃ (10 mL), and the productwas extracted with ethyl acetate (3×50 mL). Extracts were washed withbrine, dried over anhydrous Na₂SO₄, and evaporated to oil. Evaporationof fractions gave the title compound as a viscous oil in a yield of 850mg (67.6%). ³¹P NMR (mixture of diastereomers, CD₃CN): δ 148.34, 147.71.

Example 90N²-Phenoxyacetyl-5′-O-(4,4′-dimethoxytrityl)-2′-fluoro-3′-O—[(N,N-diisopropylamino)[11-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6,9-trioxaundecyl]phosphino]-2′-deoxyguanosine (59b)

Solution of bis(N,N-diisopropylamino) chlorophosphite (330 mg, 1.24mmol) in anhydrous dichloromethane (3 mL) was added dropwise to astirred solution ofN²-phenoxyacetyl-5′-O-(4,4′-dimethoxytrityl)-2′-fluoro-2′-deoxyguanosine,51b, (715 mg, 0.99 mmol) and N-ethyl-N,N-diisopropylamine (243 mg, 1.88mmol) in anhydrous dichloromethane (4 mL) at −20° C. The solution wasallowed to stir at room temperature for 2 h. To the obtained mixture,compound 7d (622 mg, 1.19 mmol) and 1H-tetrazole (0.45 M in MeCN, 0.9mL) were added followed by stirring at room temperature for 14 h. Thereaction mixture was quenched by addition of excess 5% aqueous NaHCO₃.The emulsion was diluted with 5% aqueous NaHCO₃ (10 mL), and the productwas extracted with ethyl acetate (3×50 mL). Extracts were washed withbrine, dried over anhydrous Na₂SO₄, and evaporated to oil. Evaporationof fractions gave the title compound as a viscous oil in a yield of 840mg (61.7%). ³¹P NMR (mixture of diastereomers, CD₃CN): δ 148.59, 147.90.

Example 915′-O-(4,4′-Dimethoxytrityl)-3′-O—[(N,N-diisopropylamino)[5-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3-oxapentyloxy]phosphinyl]thymidine(28)

To the magnetically stirred mixture of1-O-(5-hydroxy-3-oxapentyl)-3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranoside,7b, (501 g, 1.15 mmol) N-ethyl-N,N-diisopropylamine (282 mg, 2.19 mmol),and anhydrous CH₃CN (10 mL) was added a solution ofN,N,N′,N′-tetraisopropyl chlorophosphite (364 mg, 1.44 mmol) inanhydrous DCM (5 mL) at −20° C. The mixture was allowed to warm up toroom temperature over 2 h. A solution of5′-O-(4,4′-dimethoxytrityl)thymidine, 8, (565 mg, 1.09 mmol), inanhydrous DCM (5 mL) was added followed by the addition of a solution of1H-tetrazole in acetonitrile (0.45 M, 1.1 mL). The reaction mixture wasstirred for 18 h at room temperature and was quenched by adding 5%aqueous NaHCO₃. The product was extracted with ethyl acetate (3×50 mL).Combined extracts were washed with brine (50 mL), dried over anhydrousNa₂SO₄, and evaporated to dryness. The product was isolated by columnchromatography on silica gel eluted with a step gradient starting fromTEA—ethyl acetate—hexane (5:20:75) to a mixture of TEA in ethyl acetate(10:90). Evaporation of fractions gave compound 24 as white solid foamin a yield of 941 mg (77.7%). ³¹P NMR (mixture of diastereomers, CD₃CN):δ 148.12, 147.63.

Example 92

Oligonucleotides 61-64, 67-72, and 81-84 were synthesized by thephosphoramidite method on an Applied Biosystems DNA/RNA synthesizer 394on 1 μmol scale starting with a commercial DMT-nucleoside-succinyl-CPG(1000 Å, Glen Research, Sterling, Va.). The standard synthetic cycleuseful in assemblying oligonucleotides with phosphate backbone comprisedthe following steps:

-   -   (a) detritylation of the solid phase-bound material with 3%        dichloroacetic acid in dichloromethane followed by washing with        acetonitrile;    -   (b) coupling of nucleoside phosphoramidite building block (0.1 M        in acetonitrile) required by the sequence to the solid        support-bound material in the presence of 1H-tetrazole (0.45 M        in acetonitrile);    -   (c) capping of unreacted solid support-bound hydroxy groups with        a mixture of acetic anhydride and N-methyl imidazole and    -   (d) oxidation of the solid support-bound phosphite triester        groups with iodine (0.05 M in a mixture of pyridine,        tetrahydrofurane, water).        Preparation of oligonucleotide phosphorothioates was conducted        as disclosed in U.S. Pat. No. 7,723,528. Accordingly, steps (a)        and (b) remained as disclosed above. The iodine solution was        replaced with a solution of        N′-(3-thioxo-3H-1,2,4-dithiazol-5-yl)-N,N-dimethylmethanimidamide        (0.025 M in pyridine). Upon the completion of the coupling        step (b) and washing the solid phase with acetonitrile, the        sulfurization step was conducted. The capping step followed the        sulfurization. The cycle appropriate for the assembly of the        desired oligonucleotide was repeated as required by the sequence        in preparation.

The synthetic cycle for incorporation of the protected ribonucleosidephosphoramidite building blocks was different from that disclosed abovein that the coupling time was extended to 10 min.

The synthetic cycle for incorporation of the phosphoramidite buildingblocks disclosed in the present invention was different from thatdisclosed above in that the coupling time was extended to 1 min.

The final release of oligonucleotides 61-64 from the solid support,deprotection of internucleosidic phosphates, and monosaccharide residueswas carried out by treatment under the following conditions:

-   -   A. Concentrated ammonium hydroxide, room temperature, 18 h;    -   B. Concentrated aqueous ammonium hydroxide, 65° C., 8 h;    -   C. 0.05 M K₂CO₃ in anhydrous MeOH, room temperature, 4 h;    -   D. 0.5 M Piperidine in anhydrous acetonitrile, room temperature,        15 min, followed by a mixture of 1,2-diaminoethane and toluene        (50:50 v/v), room temperature, 4 h;    -   E. 25% Aqueous t-butylamine, 65° C., 4 h;    -   F. Mixture of concentrated aqueous ammonium hydroxide and 40%        aqueous methylamine (50:50 v/v), room temperature, 120 min.

Upon completion of the deprotection under the conditions A, B, E, and F,the liquid phase was collected and evaporated in vacuo to dryness. Theresidue was dissolved in water (1 mL) and analyzed by reverse-phase HPLCand by ES MS.

Upon completion of the deprotection under the condition C, the reactionmixtures were neutralized by addition of 0.25 M aqueous NaH₂PO₄ (250 μLper 1 mL of deprotection solution). The solid phase was spun down; theliquid phase was collected and analyzed as described above.

Upon completion of the deprotection under the condition D, the solidphase was spun down, and the liquid phase was discarded. The solid phasewas washed with toluene (2×1 mL) and with acetonitrile (2×1 mL). Thesolid phase was suspended in water (1 mL) and the suspension wascentrifugated. The liquid phase was collected and analyzed as describedabove.

The final release of oligonucleotides 67-72 and 81-84 from the solidsupport, deprotection of internucleosidic phosphates, monosaccharideresidues, and nucleic bases was carried out by treatment withconcentrated aqueous ammonium hydroxide (2 mL) at room temperature for18 h. The removal of 2′-O-tBDMS groups from oligonucleotide 84 (SEQ. ID15) was additionally carried out as disclosed in Song, Q. and Jones, R.A. Tetrahedron Lett. 1999, 40, 4653-4654, which is incorporated byreference herein in its entirety. Upon evaporation in vacuo, the crudedeprotection mixtures were dissolved in water and analyzed by ES MS andreverse-phase HPLC. HPLC analysis was carried out on Phenomenex GeminiC18 column (4.6×150 mm) using 0.05 M Tris-HCl as Buffer A, acetonitrileas Buffer B, and a linear gradient from 0 to 100% B in 45 min at a flowrate of 1 mL/min. Peaks were detected by a UV-VIS detector at 264 nm.

Skilled artisans will appreciate that numerous changes and modificationsmay be made to the preferred embodiments of the invention and that suchchanges and modifications may be made without departing from the spiritof the invention. It is therefore intended that the appended claimscover all such equivalent variations as fall within the true spirit andscope of the invention.

What is claimed is:
 1. A method of preparing a syntheticoligonucleotide, the method comprising reacting a compound of Formula T

wherein: R¹ is an optionally protected nucleic base selected from9-adeninyl, 1-cytosinyl, 9-guaninyl, 1-thyminyl, 1-uracilyl,2-amino-9-adeninyl, N⁶-methyl-9-adeninyl, 7-deaza-9-adeninyl,7-deaza-8-aza-9-adeninyl, 8-amino-9-adeninyl, 5-methyl-1-cytosinyl,N⁴-ethyl-1-cytosinyl, 7-deaza-9-guaninyl, 7-deaza-8-aza-9-guaninyl,8-amino-9-guaninyl, 7-deaza-9-xanthinyl, 9-hypoxanthinyl,N⁶-benzoyl-9-adeninyl, N⁶-phenoxyacetyl-9-adeninyl,N⁶-(4-isopropylphenoxy)acetyl-9-adeninyl, N⁴-benzoyl-1-cytosinyl,N⁴-acetyl-1-cytosinyl, N⁴-phenoxyacetyl-1-cytosinyl,N⁴-(4-isopropylphenoxy)acetyl-1-cytosinyl,N⁴-benzoyl-5-methyl-1-cytosinyl, N⁴-acetyl-5-methyl-1-cytosinyl,N⁴-phenoxyacetyl-5-methyl-1-cytosinyl,N⁴-(4-isopropylphenoxy)acetyl-5-methyl-1-cytosinyl,N²-isobutyryl-9-guaninyl, N²-phenoxyacetyl-9-guaninyl,N²-(4-isopropylphenoxy)acetyl-9-guaninyl, andN²—(N,N-dimethylformamidino)-9-guaninyl; one of R² and R^(2a) is aprotecting group of the trityl type selected from(4-methoxyphenyl)diphenylmethyl, bis-(4-methoxyphenyl)phenylmethyl,tris-(methoxyphenyl)methyl, 9-phenylxanthen-9-yl, and9-(p-methoxyphenyl)xanthen-9-yl, and the other is a phosphoramiditemoiety:

wherein: each R⁴ and R⁵ is, independently, C₁ to C₆ alkyl, or R⁴ and R⁵together with the nitrogen atom they are attached to form a cycle,wherein R⁴+R⁵═(CH₂)_(n)X(CH₂)_(m), wherein: X is an atom of oxygen orCH₂ group; each n and m is, independently, an integer from 2 to about 5;L is a linking moiety —[(CH₂)_(p)Y(CH₂)_(q)]_(r) wherein: each p, q, andr is, independently, an integer from 1 to 18; Y is a chemical bond,oxygen atom, sulfur atom, Q¹, —N(Q¹)C(═O)N(Q²)-, —C(═O)N(Q¹)-, or—N(Q¹)C(═O)—, wherein: each Q¹ and Q² is hydrogen atom, a methyl group,an ethyl group, a propyl group, an isopropyl group, an acetyl group, atrifluoroacetyl group, a phenoxyacetyl group, a benzoyl group, or a9-fluorenylmethyloxycarbonyl group; R⁶ is a substituted monosaccharideresidue of Formula II

wherein: each R¹⁰ is, independently, an acyl protecting group, atrityl-type protecting group, a silyl protecting group, an alkyl groupcontaining from 1 to 18 atoms of carbon, a benzyl group, a4-methoxybenzyl group, a propargyl group, or one of R¹⁰ is a protectedmonosaccharide residue of Formula II, and the other R¹⁰ is an acetylgroup; A is a chemical bond or CHOR¹⁰; Z is a hydrogen, OR¹⁰, orN(Q²a)Q³ wherein: each Q^(2a) and Q³ is, independently, a hydrogen atom,a methyl group, an ethyl group, a propyl group, an isopropyl group, anacetyl group, a trifluoroacetyl group, a phenoxyacetyl group, a benzoylgroup, or a 9-fluorenylmethyloxycarbonyl group; R³ is a hydrogen atom, afluorine atom, a substituted hydroxy group OR⁷, or a substituted aminogroup NR⁸R⁹ wherein: each R⁷ is, independently, a C₁ to C₆ alkyl,2-alkoxyethyl group, or an N-methylcarboxamidomethyl group; and each R⁸and R⁹ is, independently, a hydrogen atom, a methyl group, an ethylgroup, a propyl group, an isopropyl group, an acetyl group, atrifluoroacetyl group, a phenoxyacetyl group, a benzoyl group, or a9-fluorenylmethyloxycarbonyl group; with a compound of Formula IV:

wherein: one of R¹² and R¹³ is a hydrogen atom, and the other is aprotecting group or a linker connected to a solid support; k is aninteger selected from 0 to about 100; and R¹, R³, R¹¹, and W¹ have thesame values as set forth above for Formula I.
 2. The method of claim 1,wherein R¹ is a nucleic base selected from the group consisting ofN⁶-benzoyl-9-adeninyl, N⁶-phenoxyacetyl-9-adeninyl,N⁶-(4-isopropylphenoxy)acetyl-9-adeninyl, 9-adeninyl,N⁴-benzoyl-1-cytosinyl, N⁴-acetyl-1-cytosinyl,N⁴-phenoxyacetyl-1-cytosinyl, N⁴-(4-isopropylphenoxy)acetyl-1-cytosinyl,N⁴-benzoyl-5-methyl-1-cytosinyl, N⁴-acetyl-5-methyl-1-cytosinyl,1-cytosinyl, N⁴-phenoxyacetyl-5-methyl-1-cytosinyl,N⁴-(4-isopropylphenoxy)acetyl-5-methyl-1-cytosinyl,5-methyl-1-cytosinyl, N²-isobutyryl-9-guaninyl,N²-phenoxyacetyl-9-guaninyl, N²-(4-isopropylphenoxy)acetyl-9-guaninyl,N²—(N,N-dimethylformamidino)-9-guaninyl, 9-guaninyl, 1-thyminyl, and1-uracilyl.
 3. The method of claim 1, wherein R² is a4,4′-dimethoxytrityl group and R^(2a) is


4. The method of claim 1, wherein R^(2a) is a 4,4′-dimethoxytrityl groupand R² is


5. The method of claim 1, wherein R³ is a hydrogen atom.
 6. The methodof claim 1, wherein R³ is OCH₃.
 7. The method of claim 1, wherein R³ isa fluorine atom.
 8. The method of claim 1, wherein each R⁴ and R⁵ is anisopropyl group.
 9. The method of claim 1, wherein L is —(CH₂)₃—. 10.The method of claim 1, wherein L is —(CH₂)₄—.
 11. The method of claim 1,wherein L is —[(CH₂)₂—O]₂—.
 12. The method of claim 1, wherein L is—[(CH₂)₂—O]₃—.
 13. The method of claim 1, wherein L is —[(CH₂)₂—O]₄—.14. The method of claim 1, wherein L is —[(CH₂)₂—O]₅—.
 15. The method ofclaim 1, wherein R⁶ is a protected β-D-glucopyranosidyl group.
 16. Themethod of claim 1, wherein R⁶ is a protected β-D-galactopyranosidylgroup.
 17. The method of claim 1, wherein R⁶ is a protected2-amino-2-deoxy-β-D-glucopyranosidyl group.
 18. The method of claim 17,wherein one of Q^(2a) and Q³ is a hydrogen atom, and the other is anacetyl group.
 19. The method of claim 1, wherein R⁶ is a protected2-amino-2-deoxy-β-D-galactopyranosidyl group.
 20. The method of claim19, wherein one of Q^(2a) and Q³ is a hydrogen atom, and the other is anacetyl group.
 21. The method of claim 1, wherein each of R¹⁰ is anacetyl group.
 22. The method of claim 1, wherein each of R¹⁰ is abenzoyl group.
 23. The method of claim 1, wherein each of R¹⁰ is abutyryl group.
 24. The method of claim 1, wherein each of R¹⁰ is anisobutyryl group.
 25. The compound of claim 1, wherein each of R¹⁰ is apropionyl group.
 26. The method of claim 1, wherein each of R¹⁰ is a4-methylbenzoyl group.
 27. The method of claim 1, wherein one of R¹⁰ isa protected monosaccharide residue of Formula II, and the other R¹⁰ isan acetyl group.